Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

PubMed Central

Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.

2014-01-01

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411

Three novel NY-ESO-1 epitopes bound to DRB1*0803, DQB1*0401 and DRB1*0901 recognized by CD4 T cells from CHP-NY-ESO-1-vaccinated patients.

PubMed

Mizote, Yu; Taniguchi, Taku; Tanaka, Kei; Isobe, Midori; Wada, Hisashi; Saika, Takashi; Kita, Shoichi; Koide, Yukari; Uenaka, Akiko; Nakayama, Eiichi

2010-07-19

Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination. (c) 2010 Elsevier Ltd. All rights reserved.

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

PubMed

Goto, H; Minamoto, N; Ito, H; Ito, N; Sugiyama, M; Kinjo, T; Kawai, A

2000-01-01

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

Comprehensive epitope mapping of the Epstein-Barr virus latent membrane protein-2 in normal, non tumor-bearing individuals.

PubMed

Provenzano, Maurizio; Selleri, Silvia; Jin, Ping; Wang, Ena; Werden, Rosemary; Slezak, Stephanie; Adams, Sharon D; Panelli, Monica C; Leitman, Susan F; Stroncek, David F; Marincola, Francesco M

2007-07-01

Latent membrane protein (LMP)-2 is one of the Epstein-Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation.

HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers

PubMed Central

Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; LaBranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

2015-01-01

A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry.

PubMed

Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J

2006-11-01

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.

High Throughput T Epitope Mapping and Vaccine Development

PubMed Central

Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio

2010-01-01

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

PubMed

Batsuli, Glaivy; Deng, Wei; Healey, John F; Parker, Ernest T; Baldwin, W Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete; Meeks, Shannon L

2016-10-20

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. © 2016 by The American Society of Hematology.

Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

PubMed

Brier, S; Le Mignon, M; Jain, K; Lebrun, C; Peurois, F; Kellenberger, C; Bordas-Le Floch, V; Mascarell, L; Nony, E; Moingeon, P

2018-05-01

Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

PubMed

Schieferdecker, Aneta; Voigt, Mareike; Riecken, Kristoffer; Braig, Friederike; Schinke, Thorsten; Loges, Sonja; Bokemeyer, Carsten; Fehse, Boris; Binder, Mascha

2014-08-30

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins.

PubMed Central

García-Barreno, B; Palomo, C; Peñas, C; Delgado, T; Perez-Breña, P; Melero, J A

1989-01-01

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins. Images PMID:2463385

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats.

PubMed

Subramanian, Sarada; Andal, S; Karande, Anjali A; Radhakantha Adiga, P

2003-11-07

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.

Molecular determinants of T cell epitope recognition to the common Timothy grass allergen.

PubMed

Oseroff, Carla; Sidney, John; Kotturi, Maya F; Kolla, Ravi; Alam, Rafeul; Broide, David H; Wasserman, Stephen I; Weiskopf, Daniela; McKinney, Denise M; Chung, Jo L; Petersen, Arnd; Grey, Howard; Peters, Bjoern; Sette, Alessandro

2010-07-15

We investigated the molecular determinants of allergen-derived T cell epitopes in humans utilizing the Phleum pratense (Timothy grass) allergens (Phl p). PBMCs from allergic individuals were tested in ELISPOT assays with overlapping peptides spanning known Phl p allergens. A total of 43 distinct antigenic regions were recognized, illustrating the large breadth of grass-specific T cell epitopes. Th2 cytokines (as represented by IL-5) were predominant, whereas IFN-gamma, IL-10, and IL-17 were detected less frequently. Responses from specific immunotherapy treatment individuals were weaker and less consistent, yet similar in epitope specificity and cytokine pattern to allergic donors, whereas nonallergic individuals were essentially nonreactive. Despite the large breadth of recognition, nine dominant antigenic regions were defined, each recognized by multiple donors, accounting for 51% of the total response. Multiple HLA molecules and loci restricted the dominant regions, and the immunodominant epitopes could be predicted using bioinformatic algorithms specific for 23 common HLA-DR, DP, and DQ molecules. Immunodominance was also apparent at the Phl p Ag level. It was found that 52, 19, and 14% of the total response was directed to Phl p 5, 1, and 3, respectively. Interestingly, little or no correlation between Phl p-specific IgE levels and T cell responses was found. Thus, certain intrinsic features of the allergen protein might influence immunogenicity at the level of T cell reactivity. Consistent with this notion, different Phl p Ags were associated with distinct patterns of IL-5, IFN-gamma, IL-10, and IL-17 production.

Identification of a T-helper cell epitope on the rotavirus VP6 protein.

PubMed Central

Baños, D M; Lopez, S; Arias, C F; Esquivel, F R

1997-01-01

In this work, we have studied the T-helper (Th)-cell response against rotavirus, in a mouse model. Adult BALB/c mice were inoculated parenterally with porcine rotavirus YM, and the Th-cell response from spleen cells against the virus and two overlapping fragments of the major capsid protein VP6 (VP6(1-192) and VP6(171-397)) were evaluated in vitro. The Th cells recognized the YM virus and the two protein fragments, suggesting that there are at least two Th-cell epitopes on the VP6 molecule. To study the specificity of Th cells against VP6 at the clonal level, we established two Th-cell hybridomas cross-reactive for the VP6 protein of rotavirus strains YM and SA11. Both hybridomas recognized the VP6(171-397) polypeptide, and a synthetic peptide comprising the amino acids 289 to 302 (RLSFQLVRPPNMTP) of YM VP6 in the context of the major histocompatibility complex class II IEd molecule. The Th-cell hybridomas recognized rotavirus VP6 in a highly cross-reactive fashion, since they could be stimulated by eight different strains of rotavirus, including the murine rotavirus EDIM, that represent five G serotypes and at least two subgroups. The amino acid sequence of the VP6 epitope is highly conserved in most group A rotavirus strains sequenced so far. On the other hand, it was found that Th cells specific for the VP6 epitope may constitute an important proportion of the total polyclonal Th-cell response against rotavirus YM in spleen cells. These results demonstrate that VP6 can be a target for highly cross-reactive Th cells. PMID:8985366

Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins.

PubMed

Richard, Jonathan; Pacheco, Beatriz; Gohain, Neelakshi; Veillette, Maxime; Ding, Shilei; Alsahafi, Nirmin; Tolbert, William D; Prévost, Jérémie; Chapleau, Jean-Philippe; Coutu, Mathieu; Jia, Manxue; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R; Melillo, Bruno; Martin, Loïc; Tremblay, Cécile; Hahn, Beatrice H; Kaufmann, Daniel E; Wu, Xueling; Smith, Amos B; Sodroski, Joseph; Pazgier, Marzena; Finzi, Andrés

2016-10-01

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to "push" Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

PubMed Central

Pepperl, Sandra; Benninger-Döring, Gerlinde; Modrow, Susanne; Wolf, Hans; Jilg, Wolfgang

1998-01-01

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells. PMID:9765404

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

PubMed

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.

PubMed

Giang, Erick; Dorner, Marcus; Prentoe, Jannick C; Dreux, Marlène; Evans, Matthew J; Bukh, Jens; Rice, Charles M; Ploss, Alexander; Burton, Dennis R; Law, Mansun

2012-04-17

Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.

Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

PubMed

Remy, M H; Frobert, Y; Grassi, J

1995-08-01

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan.

PubMed

Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

2011-09-14

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

PubMed

Molinos-Albert, Luis M; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Rodríguez de la Concepción, Maria Luisa; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A; Contreras, F-Xabier; Floor, Martin; Cardona, Pere J; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

2017-01-13

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

PubMed Central

Keck, Zhen-yong; Xia, Jinming; Wang, Yong; Wang, Wenyan; Krey, Thomas; Prentoe, Jannick; Carlsen, Thomas; Li, Angela Ying-Jian; Patel, Arvind H.; Lemon, Stanley M.; Bukh, Jens; Rey, Felix A.; Foung, Steven K. H.

2012-01-01

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus. PMID:22511875

Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

PubMed

Ganeshan, Harini; Kusi, Kwadwo A; Anum, Dorothy; Hollingdale, Michael R; Peters, Bjoern; Kim, Yohan; Tetteh, John K A; Ofori, Michael F; Gyan, Ben A; Koram, Kwadwo A; Huang, Jun; Belmonte, Maria; Banania, Jo Glenna; Dodoo, Daniel; Villasante, Eileen; Sedegah, Martha

2016-02-01

Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays. Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens. For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous. These results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.

Natural antibody responses to the capsid protein in sera of Dengue infected patients from Sri Lanka.

PubMed

Nadugala, Mahesha N; Jeewandara, Chandima; Malavige, Gathsaurie N; Premaratne, Prasad H; Goonasekara, Charitha L

2017-01-01

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2-18 a.a), P11 (79-95 a.a) and P12 (86-101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1-40 a.a) and carboxy (70-100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39-56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.

Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model.

PubMed

Curciarello, Renata; Smaldini, Paola L; Candreva, Angela M; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A; Petruccelli, Silvana; Docena, Guillermo H

2014-01-01

Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Honda; R Wang; W Kong

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Honda, M.; Robinson, H.; Wang, R.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

The utility and limitations of current web-available algorithms to predict peptides recognized by CD4 T cells in response to pathogen infection #

PubMed Central

Chaves, Francisco A.; Lee, Alvin H.; Nayak, Jennifer; Richards, Katherine A.; Sant, Andrea J.

2012-01-01

The ability to track CD4 T cells elicited in response to pathogen infection or vaccination is critical because of the role these cells play in protective immunity. Coupled with advances in genome sequencing of pathogenic organisms, there is considerable appeal for implementation of computer-based algorithms to predict peptides that bind to the class II molecules, forming the complex recognized by CD4 T cells. Despite recent progress in this area, there is a paucity of data regarding their success in identifying actual pathogen-derived epitopes. In this study, we sought to rigorously evaluate the performance of multiple web-available algorithms by comparing their predictions and our results using purely empirical methods for epitope discovery in influenza that utilized overlapping peptides and cytokine Elispots, for three independent class II molecules. We analyzed the data in different ways, trying to anticipate how an investigator might use these computational tools for epitope discovery. We come to the conclusion that currently available algorithms can indeed facilitate epitope discovery, but all shared a high degree of false positive and false negative predictions. Therefore, efficiencies were low. We also found dramatic disparities among algorithms and between predicted IC50 values and true dissociation rates of peptide:MHC class II complexes. We suggest that improved success of predictive algorithms will depend less on changes in computational methods or increased data sets and more on changes in parameters used to “train” the algorithms that factor in elements of T cell repertoire and peptide acquisition by class II molecules. PMID:22467652

Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

PubMed

Rodriguez, M J; Sarraseca, J; Garcia, J; Sanz, A; Plana-Durán, J; Ignacio Casal, J

1997-09-01

Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Québec 807/94 (North American), were cloned and expressed in: (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein was investigated using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E. coli. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein, or at least a large fragment comprising the first 78 residues. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50-66). This region is well conserved among different isolates of European and North American origin and is the most hydrophilic region of the protein. However, this epitope, although recognized by the MAbs and many pig sera, is not useful for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire protein.

Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP).

PubMed

Tiwari, Ruby; Bhalla, Prem L; Singh, Mohan B

2009-02-02

Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay. Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120-170 and 224-244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction. Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.

Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies

PubMed Central

Wu, Jingbo; Zhang, Hui; Wang, Yuanzhi; Qiao, Jun; Chen, Chuangfu; Gao, Goege F.; Allain, Jean-Pierre; Li, Chengyao

2012-01-01

More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90. PMID:22457830

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

PubMed Central

Meeks, Shannon L.; Healey, John F.; Parker, Ernest T.; Barrow, Rachel T.

2007-01-01

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding β-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 β-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape. PMID:17848617

The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

PubMed

Smith, A M; Benjamin, D C

1991-02-15

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.

Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein.

PubMed

Kulkarni, Archana; Kurle, Swarali; Shete, Ashwini; Ghate, Manisha; Godbole, Sheela; Madhavi, Vijaya; Kent, Stephen J; Paranjape, Ramesh; Thakar, Madhuri

2017-01-01

HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm 3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm 3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288-330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection.

Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

PubMed Central

Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

2012-01-01

Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

PubMed Central

Molinos-Albert, Luis M.; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A.; Contreras, F.-Xabier; Floor, Martin; Cardona, Pere J.; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

2017-01-01

The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants. PMID:28084464

Naturally Occurring Antibodies That Recognize Linear Epitopes in the Amino Terminus of the Hepatitis C Virus E2 Protein Confer Noninterfering, Additive Neutralization

PubMed Central

Tarr, Alexander W.; Urbanowicz, Richard A.; Jayaraj, Dhanya; Brown, Richard J. P.; McKeating, Jane A.; Irving, William L.

2012-01-01

Chronic hepatitis C virus (HCV) infection can persist even in the presence of a broadly neutralizing antibody response. Various mechanisms that underpin viral persistence have been proposed, and one of the most recently proposed mechanisms is the presence of interfering antibodies that negate neutralizing responses. Specifically, it has been proposed that antibodies targeting broadly neutralizing epitopes located within a region of E2 encompassing residues 412 to 423 can be inhibited by nonneutralizing antibodies binding to a less conserved region encompassing residues 434 to 446. To investigate this phenomenon, we characterized the neutralizing and inhibitory effects of human-derived affinity-purified immunoglobulin fractions and murine monoclonal antibodies and show that antibodies to both regions neutralize HCV pseudoparticle (HCVpp) and cell culture-infectious virus (HCVcc) infection albeit with different breadths and potencies. Epitope mapping revealed the presence of overlapping but distinct epitopes in both regions, which may explain the observed differences in neutralizing phenotypes. Crucially, we failed to demonstrate any inhibition between these two groups of antibodies, suggesting that interference by nonneutralizing antibodies, at least for the region encompassing residues 434 to 446, does not provide a mechanism for HCV persistence in chronically infected individuals. PMID:22171278

Protein structure shapes immunodominance in the CD4 T cell response to yellow fever vaccination.

PubMed

Koblischke, Maximilian; Mackroth, Maria S; Schwaiger, Julia; Fae, Ingrid; Fischer, Gottfried; Stiasny, Karin; Heinz, Franz X; Aberle, Judith H

2017-08-21

The live attenuated yellow fever (YF) vaccine is a highly effective human vaccine and induces long-term protective neutralizing antibodies directed against the viral envelope protein E. The generation of such antibodies requires the help of CD4 T cells which recognize peptides derived from proteins in virus particles internalized and processed by E-specific B cells. The CD4 T helper cell response is restricted to few immunodominant epitopes, but the mechanisms of their selection are largely unknown. Here, we report that CD4 T cell responses elicited by the YF-17D vaccine are focused to hotspots of two helices of the viral capsid protein and to exposed strands and loops of E. We found that the locations of immunodominant epitopes within three-dimensional protein structures exhibit a high degree of overlap between YF virus and the structurally homologous flavivirus tick-borne encephalitis virus, although amino acid sequence identity of the epitope regions is only 15-45%. The restriction of epitopes to exposed E protein surfaces and their strikingly similar positioning within proteins of distantly related flaviviruses are consistent with a strong influence of protein structure that shapes CD4 T cell responses and provide leads for a rational design of immunogens for vaccination.

Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides

PubMed Central

Santona, Antonella; Carta, Franco; Fraghí, Peppinetta; Turrini, Franco

2002-01-01

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection. PMID:11748179

Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin (CD62P).

PubMed

Massaguer, A; Engel, P; Pérez-del-Pulgar, S; Bosch, J; Pizcueta, P

2000-08-01

P-selectin (CD62P) is an adhesion molecule expressed on the activated endothelium and activated platelets that is involved in the initial attachment of leukocytes to inflamed vascular endothelium. Blocking monoclonal antibodies (mAbs) and P-selectin-deficient mice have shown that P-selectin is a potential target in anti-inflammatory therapy. Most mAbs against P-selectin do not bind to conserved epitopes, including the ligand-binding region, since P-selectin from mammalian species shares high amino acid sequence homology. The aim of this study was to generate a novel panel of anti-P-selectin mAbs against the conserved epitopes present in several animal species. To produce these mAbs, P-selectin-deficient mice were immunized with a pre-B-cell line transfected with human P-selectin cDNA. Twelve mouse mAbs that recognize human P-selectin were obtained. Individual mAbs that bound to human, rat, mouse, rabbit and pig activated platelets were characterized by flow-cytometry, immunohistochemistry, adhesion assays and immunoprecipitation. Four of these mAbs (P-sel.KO.2.3, P-sel.KO.2.4, P-sel.KO.2.7 and P-sel.KO.2.12) cross-reacted with human, rat and mouse P-selectin. Another three mAbs (P-sel.KO.2.2, P-sel.KO.2.11 and P-sel.KO.2.12) blocked the attachment of HL60 cells to P-selectin-transfected COS cells, demonstrating that these mAbs inhibit P-selectin-mediated adhesion. MAb cross-blocking experiments showed that these three mAbs bind to very close and overlapping epitopes. An ELISA assay using mAbs P-sel.KO.2.3 and P-sel.KO.2.12 was designed to measure soluble rat, mouse and human P-selectin. These anti-P-selectin mAbs are unique since they recognize common epitopes conserved during mammalian evolution and they may be useful for studying P-selectin function in inflammatory models in various species.

16 kDa heat shock protein from heat-inactivated Mycobacterium tuberculosis is a homodimer - suitability for diagnostic applications with specific llama VHH monoclonals.

PubMed

Srivastava, Saurabh K; Ruigrok, Vincent J B; Thompson, Natalie J; Trilling, Anke K; Heck, Albert J R; van Rijn, Cees; Beekwilder, Jules; Jongsma, Maarten A

2013-01-01

The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.

Characterization of self-T-cell response and antigenic determinants of U1A protein with bone marrow-derived dendritic cells in NZB x NZW F1 mice.

PubMed

Suen, J L; Wu, C H; Chen, Y Y; Wu, W M; Chiang, B L

2001-07-01

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against nuclear intact structures, such as nucleosomes and small nuclear ribonucleoproteins (snRNPs). Autoantibodies against snRNPs are of special interest because they are detectable in the majority of SLE patients. Although the B-cell antigenic determinants have been well characterized, very limited data have been reported in regard to the T-cell epitopes of snRNPs. Furthermore, several studies have demonstrated that determination of the auto-T-cell epitopes recognized by freshly isolated T cells is difficult from unprimed lupus mice when self-antigen-pulsed B cells or macrophages are used as antigen-presenting cells (APCs) in vitro. In the present study, we showed a novel approach for determining the auto-T-cell epitopes, using bone marrow-derived dendritic cells (BMDCs) pulsed with the murine U1A protein - an immunodominant antigen of the U1 snRNPs - which is capable of activating freshly isolated T cells from unprimed (NZB x NZW) F1 (BWF1) mice in vitro. The T-cell epitope area was found to be located at the C-terminus of U1A, overlapping the T-cell epitope of human U1A that has been reported in human SLE. Identification of the autoreactive T-cell epitope(s) in snRNPs will help to elucidate how reciprocal T-B determinant spreading of snRNPs emerges in lupus. The results presented here also indicate that it is feasible to use this approach to further explore strategies to design immunotherapy for patients with lupus.

Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.

PubMed

Zhang, Xiaojie; Lee, Soo Jung; Young, Kelly Z; Josephson, David A; Geschwind, Michael D; Wang, Michael M

2014-10-02

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue. Four antibodies were raised in rabbits against two non-overlapping N-terminal NOTCH3 sequences. These reagents were used in immunohistochemical experiments to detect epitopes in post-mortem CADASIL brains (n=8), control brains, and cells overexpressing NOTCH3. To determine the biochemical nature of NOTCH3 epitopes, we used these antibodies to probe pure NOTCH3-Fc fusion proteins treated with acid, urea, guanidinium, ionic detergents, acrylamide, and thiol- and phosphorus-based reductants. All antibodies avidly stained arteries in 8 of 8 CADASIL brain samples. The most prominent staining was in degenerating media of leptomeningeal arteries and sclerotic penetrating vessels. Normal appearing vessels from control brains were not reactive. Antibodies did not react with cultured cells overexpressing NOTCH3 or with purified NOTCH3-Fc protein. Furthermore, treatment of pure protein with acid, chaotropic denaturants, alkylators, and detergents failed to unmask N-terminal NOTCH3 epitopes. Antibodies, however, recognized novel N-terminal epitopes in purified NOTCH3-Fc protein treated with three different reductants (DTT, beta-mercaptoethanol, and TCEP). We conclude that CADASIL arteries feature latent N-terminal NOTCH3 epitopes, suggesting the first evidence in vivo of NOTCH3 structural alterations. Published by Elsevier B.V.

Identification of a common antigenic site in the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus.

PubMed

Casal, J I; Rodriguez, M J; Sarraseca, J; Garcia, J; Plana-Duran, J; Sanz, A

1998-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein.

21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison's disease.

PubMed

Rottembourg, Diane; Deal, Cheri; Lambert, Marion; Mallone, Roberto; Carel, Jean-Claude; Lacroix, André; Caillat-Zucman, Sophie; le Deist, Françoise

2010-12-01

In autoimmune adrenal deficiency, autoantibodies target the 21-hydroxylase (21OH) protein. However, it is presumed that autoreactive T cells, rather than antibodies, are the main effectors of adrenal gland destruction, but their identification is still lacking. We performed a T-cell epitope mapping study using 49 overlapping 20mer peptides covering the 21OH sequence in patients with isolated Addison's disease, Autoimmune Polyendocrine Syndrome 1 and 2. IFNγ ELISPOT responses against these peptides were stronger, broader and more prevalent among patients than in controls, whatever the disease presentation. Five peptides elicited T-cell responses in patients only (68% sensitivity, 100% specificity). Blocking experiments identified IFNγ-producing cells as CD8 T lymphocytes, with two peptides frequently recognized in HLA-B8+ patients and a third one targeted in HLA-B35+ subjects. In particular, the 21OH(431-450) peptide was highly immunodominant, as it was recognized in more than 30% of patients, all carrying the HLA-B8 restriction element. This 21OH(431-450) region contained an EPLARLEL octamer (21OH(431-438)) predicted to bind to HLA-B8 with high affinity. Indeed, circulating EPLARLEL-specific CD8 T cells were detected at significant frequencies in HLA-B8+ patients but not in controls by HLA tetramer staining. This report enlightens disease-specific T-cell biomarkers and epitopes targeted in autoimmune adrenal deficiency. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeting a Cross-Reactive Gly m 5 Soy Peptide as Responsible for Hypersensitivity Reactions in a Milk Allergy Mouse Model

PubMed Central

Curciarello, Renata; Smaldini, Paola L.; Candreva, Angela M.; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A.

2014-01-01

Background Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Methods Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Results Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Conclusions Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide. PMID:24416141

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

PubMed

Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

2016-01-01

Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

Molecular basis of immunogenicity to botulinum neurotoxins and uses of the defined antigenic regions.

PubMed

Atassi, M Z

2015-12-01

Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design. Copyright © 2015 Elsevier Ltd. All rights reserved.

Catalytic domain of PDC-E2 contains epitopes recognized by antimitochondrial antibodies in primary biliary cirrhosis.

PubMed

Braun, Sandra; Berg, Christoph; Buck, Sandra; Gregor, Michael; Klein, Reinhild

2010-02-28

To search for further immunodominant peptides of the pyruvate dehydrogenase complex E2-component (PDC-E2) recognized by antimitochondrial antibodies (AMA) in primary biliary cirrhosis (PBC). Sera from 95 patients with PBC were tested by enzyme-linked immunosorbent assay against 33 synthetic overlapping peptides (25 amino acids; aa) covering the entire length of the E2-subunit of PDC-E2. Furthermore, the inner lipoyl peptide 167-184 was used in an unlipoylated and a lipoylated form as well as coupled to ovalbumin. Sera from 11 AMA negative/ANA positive PBC patients, 63 patients with other liver disorders and 22 healthy blood donors served as controls. Of the 95 PBC-sera, 74% reacted with the peptide 475-499 and 58% with the peptide 407-431 located within the catalytic domain of PDC-E2. Patients with other disorders or healthy controls were positive in only up to 18%. Antibodies to the unlipoylated and lipoylated peptide 167-184 within the inner lipoyl domain were found in only 5% and 11% of the PBC sera, respectively; using ovalbumin-coupled peptides, the incidence increased up to 57% (unlipoylated form). Peptides within the catalytic site of PDC-E2 rather than the previously reported lipoyl binding peptide 167-184 may represent major immunodominant epitopes recognized by AMA in PBC.

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes

PubMed Central

Klein, R; Zanger, U M; Berg, T; Hopf, U; Berg, P A

1999-01-01

Anti-liver-kidney microsome antibodies (anti-LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti-LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti-LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti-LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti-LKM-positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254–339 (254–273, 257–269, 270–294, 291–310, 307–324, 321–339, 373–389) as well as the novel peptide 196–218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6-dependent bufuralol 1′-hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257–269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196–218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254–273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti-LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti-LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257–269 a further immunodominant epitope exists on CYP2D6 which is recognized by sera from patients with AIH II but hardly by sera from patients with hepatitis C. PMID:10540193

Isotypes and antigenic profiles of pemphigus foliaceus and pemphigus vulgaris autoantibodies.

PubMed

Hacker, Mary K; Janson, Marleen; Fairley, Janet A; Lin, Mong-Shang

2002-10-01

In this study we systematically characterized isotype profiles and antigenic and tissue specificity of antidesmoglein autoantibodies from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) using enzyme-linked immunoabsorbent assays (ELISA), indirect immunofluorescence (IIF) staining, and immunoblotting (IB). In PF, we found that IgG1 antidesmoglein-1 (Dsg1) reacts with a linear epitope(s) on the ectodomain of Dsg1, while its IgG4 counterpart recognizes a conformational epitope(s). These two subclasses of anti-Dsg1 are both capable of recognizing tissues from monkey esophagus and adult human skin, but IgG1 is not able to react with mouse skin, which may explain why this isotype of anti-Dsg1 failed to induce PF-like lesions in the passive transfer animal model. In mucosal PV patients, we found that both IgG1 and IgG4 only recognized monkey esophagus tissue by IIF, except in one patient, indicating that these antibodies react with a unique conformational epitope(s) that is present in mucosal but not skin tissue. In generalized PV, IgG1 anti-Dsg3 autoantibodies appeared to recognize a linear epitope(s) on the Dsg3 ectodomain. In contrast, IgG4 anti-Dsg3 antibodies recognized both linear and conformational epitopes on the Dsg3 molecule. Interestingly, the IgG1 anti-Dsg3 antibodies failed to react with human and mouse skin tissues, suggesting that this subclass of autoantibodies may not play an essential role in the development of PV suprabasilar lesions. In summary, we conclude that this study further elucidates the pathological mechanisms of PF and PV autoantibodies by revealing their distinct isotype and antigenic profiles. This information may help us to better understand the autoimmune mechanisms underlying the development of pemphigus.

Identification of novel rabbit hemorrhagic disease virus B-cell epitopes and their interaction with host histo-blood group antigens.

PubMed

Song, Yanhua; Wang, Fang; Fan, Zhiyu; Hu, Bo; Liu, Xing; Wei, Houjun; Xue, Jiabin; Xu, Weizhong; Qiu, Rulong

2016-02-01

Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.

Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine.

PubMed

He, Yuxian; Li, Jingjing; Du, Lanying; Yan, Xuxia; Hu, Guangan; Zhou, Yusen; Jiang, Shibo

2006-06-29

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.

A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects

PubMed Central

Simon-Vecsei, Zsófia; Király, Róbert; Bagossi, Péter; Tóth, Boglárka; Dahlbom, Ingrid; Caja, Sergio; Csősz, Éva; Lindfors, Katri; Sblattero, Daniele; Nemes, Éva; Mäki, Markku; Fésüs, László; Korponay-Szabó, Ilma R.

2012-01-01

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1–2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits. PMID:22198767

Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

PubMed Central

Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

2012-01-01

Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes simplex virus 2 glycoprotein D.

PubMed

Whitbeck, J Charles; Huang, Zhen-Yu; Cairns, Tina M; Gallagher, John R; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B; Eisenberg, Roselyn J; Cohen, Gary H

2014-07-01

The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366: 34-43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. Importance: Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2). Although several distinct, well-characterized virus-neutralizing epitopes on gD2 are targeted by murine monoclonal antibodies, it is not known whether the same epitopes are targeted by the humoral response to gD2 in humans. We have developed a novel, biosensor-based competition assay to directly address this important question. Using this approach, we identified epitopes that elicit strong humoral responses in humans, as well as other epitopes that elicit much weaker responses. These data provide new insight into the human response to known neutralizing gD2 epitopes and reveal characteristics of this response that may guide future vaccine development. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

PubMed Central

Huang, Zhen-Yu; Cairns, Tina M.; Gallagher, John R.; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B.; Eisenberg, Roselyn J.; Cohen, Gary H.

2014-01-01

ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2). Although several distinct, well-characterized virus-neutralizing epitopes on gD2 are targeted by murine monoclonal antibodies, it is not known whether the same epitopes are targeted by the humoral response to gD2 in humans. We have developed a novel, biosensor-based competition assay to directly address this important question. Using this approach, we identified epitopes that elicit strong humoral responses in humans, as well as other epitopes that elicit much weaker responses. These data provide new insight into the human response to known neutralizing gD2 epitopes and reveal characteristics of this response that may guide future vaccine development. PMID:24789783

Kinetics of HIV-1 CTL epitopes recognized by HLA I alleles in HIV-infected individuals at times near primary infection: the Provir/Latitude45 study.

PubMed

Papuchon, Jennifer; Pinson, Patricia; Guidicelli, Gwenda-Line; Bellecave, Pantxika; Thomas, Réjean; LeBlanc, Roger; Reigadas, Sandrine; Taupin, Jean-Luc; Baril, Jean Guy; Routy, Jean Pierre; Wainberg, Mark; Fleury, Hervé

2014-01-01

In patients responding successfully to ART, the next therapeutic step is viral cure. An interesting strategy is antiviral vaccination, particularly involving CD8 T cell epitopes. However, attempts at vaccination are dependent on the immunogenetic background of individuals. The Provir/Latitude 45 project aims to investigate which CTL epitopes in proviral HIV-1 will be recognized by the immune system when HLA alleles are taken into consideration. A prior study (Papuchon et al, PLoS ONE 2013) showed that chronically-infected patients under successful ART exhibited variations of proviral CTL epitopes compared to a reference viral strain (HXB2) and that a generic vaccine may not be efficient. Here, we investigated viral and/or proviral CTL epitopes at different time points in recently infected individuals of the Canadian primary HIV infection cohort and assessed the affinity of these epitopes for HLA alleles during the study period. An analysis of the results confirms that it is not possible to fully predict which epitopes will be recognized by the HLA alleles of the patients if the reference sequences and epitopes are taken as the basis of simulation. Epitopes may be seen to vary in circulating RNA and proviral DNA. Despite this confirmation, the overall variability of the epitopes was low in these patients who are temporally close to primary infection.

Kinetics of HIV-1 CTL Epitopes Recognized by HLA I Alleles in HIV-Infected Individuals at Times near Primary Infection: The Provir/Latitude45 Study

PubMed Central

Papuchon, Jennifer; Pinson, Patricia; Guidicelli, Gwenda-Line; Bellecave, Pantxika; Thomas, Réjean; LeBlanc, Roger; Reigadas, Sandrine; Taupin, Jean-Luc; Baril, Jean Guy; Routy, Jean Pierre; Wainberg, Mark; Fleury, Hervé

2014-01-01

In patients responding successfully to ART, the next therapeutic step is viral cure. An interesting strategy is antiviral vaccination, particularly involving CD8 T cell epitopes. However, attempts at vaccination are dependent on the immunogenetic background of individuals. The Provir/Latitude 45 project aims to investigate which CTL epitopes in proviral HIV-1 will be recognized by the immune system when HLA alleles are taken into consideration. A prior study (Papuchon et al, PLoS ONE 2013) showed that chronically-infected patients under successful ART exhibited variations of proviral CTL epitopes compared to a reference viral strain (HXB2) and that a generic vaccine may not be efficient. Here, we investigated viral and/or proviral CTL epitopes at different time points in recently infected individuals of the Canadian primary HIV infection cohort and assessed the affinity of these epitopes for HLA alleles during the study period. An analysis of the results confirms that it is not possible to fully predict which epitopes will be recognized by the HLA alleles of the patients if the reference sequences and epitopes are taken as the basis of simulation. Epitopes may be seen to vary in circulating RNA and proviral DNA. Despite this confirmation, the overall variability of the epitopes was low in these patients who are temporally close to primary infection. PMID:24964202

Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.

PubMed

Sharma, Girdhari M; Irsigler, Andre; Dhanarajan, Pushparani; Ayuso, Rosalia; Bardina, Luda; Sampson, Hugh A; Roux, Kenneth H; Sathe, Shridhar K

2011-04-27

Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

PubMed

Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

2010-10-01

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Analysis of the epitope structure of Plum pox virus coat protein.

PubMed

Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

2011-05-01

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

Identification of epitopes of the β subunit of soybean β-conglycinin that are antigenic in pigs, dogs, rabbits and fish.

PubMed

Taliercio, Earl; Loveless, Telisa M; Turano, Marc J; Kim, Sung Woo

2014-08-01

β-Conglycinin (conglycinin) is one of the major seed storage proteins of soybean. Conglycinin is a 7S trimer composed of different combinations of β, α and α' subunits. All subunits of conglycinin have been reported to be allergenic in humans. The goal of this research is to identify epitopes of the β subunit of conglycinin that are antigenic in multiple animal species. Sera from pigs, dogs, rabbits and hybrid striped bass that had antibodies against soybean conglycinin were identified by ELISA. Most of these sera recognized peptides that represent the β subunit of conglycinin. One antigenic region of the β subunit of conglycinin had considerable overlap among all species tested. One region that was similar to a peanut allergenic epitope in humans overlapped with a region that binds IgE from dogs. One region was antigenic in multiple rabbits and pigs, suggesting it may play a role in the response of pigs to soybean in the diet. One region of the β subunit of conglycinin is an important antigen across species and abuts a region similar to the peanut allergen ARA h 1. A second region is particularly antigenic in pigs and rabbits. Variants of these antigenic regions of the β subunit of conglycinin may be useful in determining the role these regions play in the health of animals fed soybean. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients*

PubMed Central

Hecker, Michael; Fitzner, Brit; Wendt, Matthias; Lorenz, Peter; Flechtner, Kristin; Steinbeck, Felix; Schröder, Ina; Thiesen, Hans-Jürgen; Zettl, Uwe Klaus

2016-01-01

Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g. MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (p values <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392–411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS. PMID:26831522

Immune Control of Burkholderia pseudomallei––Common, High-Frequency T-Cell Responses to a Broad Repertoire of Immunoprevalent Epitopes

PubMed Central

Nithichanon, Arnone; Rinchai, Darawan; Buddhisa, Surachat; Saenmuang, Pornpun; Kewcharoenwong, Chidchamai; Kessler, Bianca; Khaenam, Prasong; Chetchotisakd, Ploenchan; Maillere, Bernard; Robinson, John; Reynolds, Catherine J.; Boyton, Rosemary J.; Altmann, Daniel M.; Lertmemongkolchai, Ganjana

2018-01-01

Burkholderia pseudomallei (Bp) is an environmental bacterial pathogen that causes potentially lethal sepsis in susceptible individuals and is considered a Category B, Tier-1 biothreat agent. As such, it is crucial to gain an improved understanding of protective immunity and potential vaccine candidates. The nature of immune correlates dictating why most exposed individuals in endemic regions undergo asymptomatic seroconversion while others succumb to life-threatening sepsis is largely uncharted. Bp seroreactive, immunogenic proteins have previously been identified by antigen microarray. We here set out to conduct an analysis of T-cell recognition of the Bp immunome using serodominant antigens represented in the original antigen microarray, examining immune correlates of disease in healthy seropositive individuals and those with acute disease or in convalescence. By screening a library of 739 overlapping peptides representing the sequences of 20 different Bp antigens, we aimed to define immune correlates of protection at the level of immunoprevalent T-cell epitopes. Responses to a large number of epitopes were common in healthy seropositive individuals: we found remarkably broad responsiveness to Bp epitopes, with 235 of 739 peptides recognized by ≥80% of all tested donors. The cumulative response to Bp epitopes in healthy, seropositive, donors from this endemic region were of the order of thousands of spot forming cells per million cells, making Bp recognition a significant component of the T-cell repertoire. Noteworthy among our findings, analysis revealed 10 highly immunoprevalent T-cell epitopes, able to induce Bp-specific IFNγ responses that were high in responding T-cell frequency within the repertoire, and also common across individuals with different human leukocyte antigen types. Acute melioidosis patients showed poor T-cell responses to the immunoprevalent epitopes, but acquired responsiveness following recovery from infection. Our findings suggest that a large repertoire of CD4 T cells, high in frequency and with broad coverage of antigens and epitopes, is important in controlling Bp infection. This offers an attractive potential strategy for subunit or epitope-based vaccines. PMID:29616023

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

PubMed

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M; Ludwig, Ralf J; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera ( N  = 32, with positive reactivity with CXCR3) to healthy controls ( N  = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients ( N  = 31) from normal healthy controls ( N  = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3

PubMed Central

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M.; Ludwig, Ralf J.; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients. PMID:29623076

Epitope analysis of the malaria surface antigen pfs48/45 identifies a subdomain that elicits transmission blocking antibodies.

PubMed

Outchkourov, Nikolay; Vermunt, Adriaan; Jansen, Josephine; Kaan, Anita; Roeffen, Will; Teelen, Karina; Lasonder, Edwin; Braks, Anneke; van de Vegte-Bolmer, Marga; Qiu, Li Yan; Sauerwein, Robert; Stunnenberg, Hendrik G

2007-06-08

Pfs48/45, a member of a Plasmodium-specific protein family, displays conformation-dependent epitopes and is an important target for malaria transmission-blocking (TB) immunity. To design a recombinant Pfs48/45-based TB vaccine, we analyzed the conformational TB epitopes of Pfs48/45. The Pfs48/45 protein was found to consist of a C-terminal six-cysteine module recognized by anti-epitope I antibodies, a middle four-cysteine module recognized by anti-epitopes IIb and III, and an N-terminal module recognized by anti-epitope V antibodies. Refolding assays identified that a fragment of 10 cysteines (10C), comprising the middle four-cysteine and the C-terminal six-cysteine modules, possesses superior refolding capacity. The refolded and partially purified 10C conformer elicited antibodies in mice that targeted at least two of the TB epitopes (I and III). The induced antibodies could block the fertilization of Plasmodium falciparum gametes in vivo in a concentration-dependent manner. Our results provide important insight into the structural organization of the Pfs48/45 protein and experimental support for a Pfs48/45-based subunit vaccine.

Definition and characterization of novel HLA-*A02-restricted CD8+ T cell epitopes derived from JCV polyomavirus with clinical relevance

PubMed Central

Mani, Jiju; Wang, Lei; Hückelhoven, Angela G.; Schmitt, Anita; Gedvilaite, Alma; Jin, Nan; Kleist, Christian; Ho, Anthony D; Schmitt, Michael

2017-01-01

Human JC and BK polyomaviruses (JCV/BKV) can establish a latent infection without any clinical symptoms in healthy individuals. In immunocompromised hosts infection or reactivation of JCV and BKV can cause lethal progressive multifocal leukoencephalopathy (PML) and hemorrhagic cystitis, respectively. Vaccination with JCV/BKV derived antigen epitope peptides or adoptive transfer of virus-specific T cells would constitute an elegant approach to clear virus-infected cells. Furthermore, donor leukocyte infusion (DLI) is another therapeutic approach which could be helpful for patients with JCV/BKV infections. So far, only few immunodominant T cell epitopes of JCV and BKV have been described and therefore is a fervent need for the definition of novel epitopes. In this study, we identified novel T cell epitopes by screening libraries of overlapping peptides derived from the major capsid protein VP1 of JCV. Virus like particles (VLPs) were used to confirm naturally processing. Two human leucocyte antigen (HLA)-A*02-restricted epitopes were characterized by fine mapping with overlapping peptides and nonamer peptide sequences were identified. Cytokine release profile of the epitope-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assays and by flow cytometry. We demonstrated that T cell responses were of polyfunctional nature with the potential of epitope-specific killing and cross-reactivity between JCV and BKV. These novel epitopes might constitute a new potential tool to design effective diagnostic and therapeutic approaches against both polyomaviruses. PMID:27705933

Definition and characterization of novel HLA-*A02-restricted CD8+ T cell epitopes derived from JCV polyomavirus with clinical relevance.

PubMed

Mani, Jiju; Wang, Lei; Hückelhoven, Angela G; Schmitt, Anita; Gedvilaite, Alma; Jin, Nan; Kleist, Christian; Ho, Anthony D; Schmitt, Michael

2017-01-10

Human JC and BK polyomaviruses (JCV/BKV) can establish a latent infection without any clinical symptoms in healthy individuals. In immunocompromised hosts infection or reactivation of JCV and BKV can cause lethal progressive multifocal leukoencephalopathy (PML) and hemorrhagic cystitis, respectively. Vaccination with JCV/BKV derived antigen epitope peptides or adoptive transfer of virus-specific T cells would constitute an elegant approach to clear virus-infected cells. Furthermore, donor leukocyte infusion (DLI) is another therapeutic approach which could be helpful for patients with JCV/BKV infections.So far, only few immunodominant T cell epitopes of JCV and BKV have been described and therefore is a fervent need for the definition of novel epitopes. In this study, we identified novel T cell epitopes by screening libraries of overlapping peptides derived from the major capsid protein VP1 of JCV. Virus like particles (VLPs) were used to confirm naturally processing. Two human leucocyte antigen (HLA)-A*02-restricted epitopes were characterized by fine mapping with overlapping peptides and nonamer peptide sequences were identified. Cytokine release profile of the epitope-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assays and by flow cytometry. We demonstrated that T cell responses were of polyfunctional nature with the potential of epitope-specific killing and cross-reactivity between JCV and BKV. These novel epitopes might constitute a new potential tool to design effective diagnostic and therapeutic approaches against both polyomaviruses.

Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum.

PubMed Central

Zheng, X; Lau, K; Frazier, M; Cassell, G H; Watson, H L

1996-01-01

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. PMID:8914774

Identification and characterization of B-cell epitopes in the DBL4ε domain of VAR2CSA.

PubMed

Ditlev, Sisse B; Nielsen, Morten A; Resende, Mafalda; Agerbæk, Mette Ø; Pinto, Vera V; Andersen, Pernille H; Magistrado, Pamela; Lusingu, John; Dahlbäck, Madeleine; Theander, Thor G; Salanti, Ali

2012-01-01

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus.

PubMed

Martínez-Torrecuadrada, J L; Langeveld, J P; Venteo, A; Sanz, A; Dalsgaard, K; Hamilton, W D; Meloen, R H; Casal, J I

1999-05-10

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes. Copyright 1999 Academic Press.

Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells

PubMed Central

1988-01-01

Immunization with the autoantigen myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE). Initial investigations indicated that encephalitogenic murine determinants of MBP were located only within MBP 1-37 and MBP 89-169. Encephalitogenic T cell epitopes within these fragments have been identified. Each epitope is recognized by T cells in association with separate allelic I-A molecules. A hybrid I-E-restricted T cell clone that recognizes intact mouse (self) MBP has been examined. The epitope recognized by this clone includes MBP residues 35-47. When tested in vivo, p35-47 causes EAE. T cell recognition of p35-47 occurs only in association with I-E molecules. These results provide the first clear example that antigen-specific T cells restricted by I-E class II molecules participate in murine autoimmune disease. Furthermore, it is clear that there are multiple (at least three) discrete encephalitogenic T cell epitopes of this autoantigen, each recognized in association with separate allelic class II molecules. These results may be relevant to human autoimmune diseases whose susceptibility is associated with more than one HLA-D molecule. PMID:2459291

Analysis of Linear Antibody Epitopes on Factor H and CFHR1 Using Sera of Patients with Autoimmune Atypical Hemolytic Uremic Syndrome.

PubMed

Trojnár, Eszter; Józsi, Mihály; Uray, Katalin; Csuka, Dorottya; Szilágyi, Ágnes; Milosevic, Danko; Stojanović, Vesna D; Spasojević, Brankica; Rusai, Krisztina; Müller, Thomas; Arbeiter, Klaus; Kelen, Kata; Szabó, Attila J; Reusz, György S; Hyvärinen, Satu; Jokiranta, T Sakari; Prohászka, Zoltán

2017-01-01

In autoimmune atypical hemolytic uremic syndrome (aHUS), the complement regulator factor H (FH) is blocked by FH autoantibodies, while 90% of the patients carry a homozygous deletion of its homolog complement FH-related protein 1 (CFHR1). The functional consequence of FH-blockade is widely established; however, the molecular basis of autoantibody binding and the role of CFHR1 deficiency in disease pathogenesis are still unknown. We performed epitope mapping of FH to provide structural insight in the autoantibody recruitment on FH and potentially CFHR1. Eight anti-FH positive aHUS patients were enrolled in this study. With overlapping synthetic FH and CFHR1 peptides, we located the amino acids (aa) involved in binding of acute and convalescence stage autoantibodies. We confirmed the location of the mapped epitopes using recombinant FH domains 19-20 that carried single-aa substitutions at the suspected antibody binding sites in three of our patients. Location of the linear epitopes and the introduced point mutations was visualized using crystal structures of the corresponding domains of FH and CFHR1. We identified three linear epitopes on FH (aa1157-1171; aa1177-1191; and aa1207-1226) and one on CFHR1 (aa276-290) that are recognized both in the acute and convalescence stages of aHUS. We observed a similar extent of autoantibody binding to the aHUS-specific epitope aa1177-1191 on FH and aa276-290 on CFHR1, despite seven of our patients being deficient for CFHR1. Epitope mapping with the domain constructs validated the location of the linear epitopes on FH with a distinct autoantibody binding motif within aa1183-1198 in line with published observations. According to the results, the linear epitopes we identified are located close to each other on the crystal structure of FH domains 19-20. This tertiary configuration contains the amino acids reported to be involved in C3b and sialic acid binding on the regulator, which may explain the functional deficiency of FH in the presence of autoantibodies. The data we provide identify the exact structures involved in autoantibody recruitment on FH and confirm the presence of an autoantibody binding epitope on CFHR1.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

PubMed

Groh, N; von Loetzen, C S; Subbarayal, B; Möbs, C; Vogel, L; Hoffmann, A; Fötisch, K; Koutsouridou, A; Randow, S; Völker, E; Seutter von Loetzen, A; Rösch, P; Vieths, S; Pfützner, W; Bohle, B; Schiller, D

2017-05-01

Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G 4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG 4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG 4 antibodies are developed is under debate. We sought to analyze the epitope specificities of IgE and IgG 4 antibodies from sera of patients who received AIT. 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG 4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a _11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG 4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. rBet v 1a _11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a _11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC 50 for IgE-mediated mediator release induced by rBet v 1a _11x was determined. The reduction of IgE and IgG 4 binding to rBet v 1a _11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG 4 to 66.1% and 64.9%, respectively. Serum IgE and IgG 4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. Patients receiving AIT develop Bet v 1a-specific IgG 4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG 4 might stimulate the development of epitope-specific diagnostics and therapeutics. © 2016 John Wiley & Sons Ltd.

[The preliminary analysis of the recognition epitopes of anti-HEV monoclonal antibodies on HEV ORF2].

PubMed

Xiong, Jun-Hui; Guo, Qing-Shun; Ge, Sheng-Xiang; Gu, Ying; Chen, Yi-Xin; Miao, Ji; Du, Hai-Lian; Shi, Wei-Guo; Zhang, Jun; Xia, Ning-Shao

2008-06-01

Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.

Towards Defining Molecular Determinants Recognized by Adaptive Immunity in Allergic Disease: An Inventory of the Available Data

PubMed Central

Vaughan, Kerrie; Greenbaum, Jason; Kim, Yohan; Vita, Randi; Chung, Jo; Peters, Bjoern; Broide, David; Goodman, Richard; Grey, Howard; Sette, Alessandro

2010-01-01

Adaptive immune responses associated with allergic reactions recognize antigens from a broad spectrum of plants and animals. Herein a meta-analysis was performed on allergy-related data from the immune epitope database (IEDB) to provide a current inventory and highlight knowledge gaps and areas for future work. The analysis identified over 4,500 allergy-related epitopes derived from 270 different allergens. Overall, the distribution of the data followed expectations based on the nature of allergic responses. Namely, the majority of epitopes were defined for B cells/antibodies and IgE-mediated reactivity, and relatively fewer T-cell epitopes, mostly CD4+/class II. Interestingly, the majority of food allergen epitopes were B-cells epitopes whereas a fairly even number of B- and T-cell epitopes were defined for airborne allergens. In addition, epitopes from nonhumans hosts were mostly T-cell epitopes. Overall, coverage of known allergens is sparse with data available for only ~17% of all allergens listed by the IUIS database. Thus, further research would be required to provide a more balanced representation across different allergen categories. Furthermore, inclusion of nonpeptidic epitopes in the IEDB also allows for inventory and analysis of immunological data associated with drug and contact allergen epitopes. Finally, our analysis also underscores that only a handful of epitopes have thus far been investigated for their immunotherapeutic potential. PMID:21403821

Differential Recognition of Mycobacterium tuberculosis-Specific Epitopes as a Function of Tuberculosis Disease History.

PubMed

Scriba, Thomas J; Carpenter, Chelsea; Pro, Sebastian Carrasco; Sidney, John; Musvosvi, Munyaradzi; Rozot, Virginie; Seumois, Grégory; Rosales, Sandy L; Vijayanand, Pandurangan; Goletti, Delia; Makgotlho, Edward; Hanekom, Willem; Hatherill, Mark; Peters, Bjoern; Sette, Alessandro; Arlehamn, Cecilia S Lindestam

2017-09-15

Individuals with a history of tuberculosis (TB) disease are at elevated risk of disease recurrence. The underlying cause is not known, but one explanation is that previous disease results in less-effective immunity against Mycobacterium tuberculosis (Mtb). We hypothesized that the repertoire of Mtb-derived epitopes recognized by T cells from individuals with latent Mtb infection differs as a function of previous diagnosis of active TB disease. T-cell responses to peptide pools in samples collected from an adult screening and an adolescent validation cohort were measured by IFN-γ enzyme-linked immunospot assay or intracellular cytokine staining. We identified a set of "type 2" T-cell epitopes that were recognized at 10-fold-lower levels in Mtb-infected individuals with a history of TB disease less than 6 years ago than in those without previous TB. By contrast, "type 1" epitopes were recognized equally well in individuals with or without previous TB. The differential epitope recognition was not due to differences in HLA class II binding, memory phenotypes, or gene expression in the responding T cells. Instead, "TB disease history-sensitive" type 2 epitopes were significantly (P < 0.0001) more homologous to sequences from bacteria found in the human microbiome than type 1 epitopes. Preferential loss of T-cell reactivity to Mtb epitopes that are homologous to bacteria in the microbiome in persons with previous TB disease may reflect long-term effects of antibiotic TB treatment on the microbiome.

Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5)

NASA Astrophysics Data System (ADS)

Zhang, Wenshuai; Zeng, Xiaoyan; Zhang, Li; Peng, Haiyan; Jiao, Yongjun; Zeng, Jun; Treutlein, Herbert R.

2013-06-01

In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

Identification of HLA-DRB1*1501-restricted T-cell epitopes from human prostatic acid phosphatase.

PubMed

Klyushnenkova, Elena N; Kouiavskaia, Diana V; Kodak, James A; Vandenbark, Arthur A; Alexander, Richard B

2007-07-01

The crucial role of CD4 T-cells in anti-tumor immune response is widely recognized, yet the identification of HLA class II-restricted epitopes derived from tumor antigens has lagged behind compared to class I epitopes. This is particularly true for prostate cancer. Based on the hypothesis that successful cancer immunotherapy will likely resemble autoimmunity, we searched for the CD4 T-cell epitopes derived from prostatic proteins that are restricted by human leukocyte antigen (HLA)-DRB1*1501, an allele associated with granulomatous prostatitis (GP), a disease that may have an autoimmune etiology. One of the antigens implicated in the development of autoimmunity in the prostate is prostatic acid phosphatase (PAP), which is also considered a promising target for prostate cancer immunotherapy. We immunized transgenic (tg) mice engineered to express HLA-DRB1*1501 with human PAP. A library of overlapping 20-mer peptides spanning the entire human PAP sequence was screened in vitro for T-cell recognition by proliferative and interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. We identified two 20-mer peptides, PAP (133-152), and PAP (173-192), that were immunogenic and naturally processed from whole PAP in HLA-DRB1*1501 tg mice. These peptides were also capable of stimulating CD4 T lymphocytes from HLA-DRB1*1501-positive patients with GP and normal donors. These peptides can be used for the design of a new generation of peptide-based vaccines against prostate cancer. The study can also be helpful in understanding the role of autoimmunity in the development of some forms of chronic prostatitis.

A combination of two antibodies recognizing non-overlapping epitopes of HER2 induces kinase activity-dependent internalization of HER2.

PubMed

Szymanska, Monika; Fosdahl, Anne M; Nikolaysen, Filip; Pedersen, Mikkel W; Grandal, Michael M; Stang, Espen; Bertelsen, Vibeke

2016-10-01

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down-regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody-induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non-overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody-induced HER2 down-regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

PubMed

Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein

PubMed Central

1992-01-01

The immunogenicity of a chimeric T/B cell peptide corresponding to antigenically characterized epitopes of the Chlamydia trachomatis major outer membrane protein (MOMP) was studied in mice to further define its potential use in the development of a subunit vaccine in preventing blinding trachoma in humans. The chimeric peptide, designated A8-VDI, corresponds to a conserved MOMP T helper (Th) cell epitope(s) (A8, residues 106-130) and serovar A VDI (residues 66-80), which contains the serovar-specific neutralizing epitope 71VAGLEK76. Mice immunized with peptide A8-VDI produced high-titered polyclonal IgG antibodies which recognized the VAGLEK-neutralizing epitope. Peptide A8-VDI primed A/J mice to produce high-titered serum-neutralizing antibodies in response to a secondary immunization with intact chlamydial elementary bodies (EBs). Peptide A8-VDI, but not peptide VDI alone, was immunogenic in six different inbred strains of mice disparate at H-2, indicating that the Th cell epitope(s) contained in the A8 portion of the chimera was recognized in the context of multiple major histocompatibility complex (MHC) haplotypes. An unexpected finding of this work was that different inbred strains of mice immunized with the chimeric peptide produced antibodies of differing fine specificities to the VDI portion of the chimera. Some mouse strains produced anti-VDI antibodies that did not recognize the VAGLEK-neutralizing epitope. The ability of mice to respond to the VAGLEK-neutralizing site was not dependent on MHC haplotype since mouse strains of the same H-2 haplotype produced anti-VDI antibodies of differing fine specificity. PMID:1370528

Artificial-epitope mapping for CK-MB assay.

PubMed

Tai, Dar-Fu; Ho, Yi-Fang; Wu, Cheng-Hsin; Lin, Tzu-Chieh; Lu, Kuo-Hao; Lin, Kun-Shian

2011-06-07

A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gromowski, Gregory D.; Barrett, Alan D.T.

2007-09-30

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity ofmore » each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.« less

Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.

PubMed

Farboud, B; Aotaki-Keen, A; Miyata, T; Hjelmeland, L M; Handa, J T

1999-07-14

To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

PubMed

Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern

2014-04-08

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Comparison of Epitope Repertoires Associated with Myasthenia Gravis in Humans and Nonhuman Hosts

PubMed Central

Vaughan, Kerrie; Kim, Yohan; Sette, Alessandro

2012-01-01

Here we analyzed the molecular targets associated with myasthenia gravis (MG) immune responses, enabled by an immune epitope database (IEDB) inventory of approximately 600 MG-related epitopes derived from 175 references. The vast majority of epitopes were derived from the α-subunit of human AChR suggesting that other MG-associated autoantigens should be investigated further. Human α-AChR was mostly characterized in humans, whereas reactivity primarily to T. californica AChR was examined in animal models. While the fine specificity of T-cell response was similar in the two systems, substantial antibody reactivity to the C-terminus was detected in the nonhuman system, but not in humans. Further analysis showed that the reactivity of nonhuman hosts to the C-terminus was eliminated when data were restricted to hosts tested in the context of autoimmune disease (spontaneous or induced), demonstrating that the epitopes recognized in humans and animals were shared when disease was present. Finally, we provided data subsets relevant to particular applications, including those associated with HLA typing or restriction, sets of epitopes recognized by monoclonal antibodies, and epitopes associated with modulation of immunity or disease. In conclusion, this analysis highlights gaps, differences, and similarities in the epitope repertoires of humans and animal models. PMID:23243503

Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2.

PubMed

Braendstrup, Peter; Mortensen, Bo Kok; Justesen, Sune; Osterby, Thomas; Rasmussen, Michael; Hansen, Andreas Martin; Christiansen, Claus Bohn; Hansen, Morten Bagge; Nielsen, Morten; Vindeløv, Lars; Buus, Søren; Stryhn, Anette

2014-01-01

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

PubMed Central

Braendstrup, Peter; Mortensen, Bo Kok; Justesen, Sune; Østerby, Thomas; Rasmussen, Michael; Hansen, Andreas Martin; Christiansen, Claus Bohn; Hansen, Morten Bagge; Nielsen, Morten; Vindeløv, Lars; Buus, Søren; Stryhn, Anette

2014-01-01

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy. PMID:24760079

Structural basis for the antibody neutralization of Herpes simplex virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Cheng-Chung; Lin, Li-Ling; Academia Sinica, Taipei 115, Taiwan

2013-10-01

The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317more » Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.« less

Mapping of IgE and IgG4 antibody-binding epitopes in Cyn d 1, the major allergen of Bermuda grass pollen.

PubMed

Yuan, Han-Chih; Wu, Keh-Gong; Chen, Chun-Jen; Su, Song-Nan; Shen, Horng-Der; Chen, Yann-Jang; Peng, Ho-Jen

2012-01-01

Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG(4) antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG(1-4) antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. Four major IgE-binding epitopes (peptides 15(-1), 21, 33(-2) and 35(+1), corresponding to amino acids 70-79, 101-110, 159-167 and 172-181) and 5 major IgG(4)-binding epitopes (peptides 15(-1), 30(-2), 33(-2), 35(+1) and 39, corresponding to amino acids 70-79, 144-153, 159-167, 172-181 and 192-200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG(4). Their critical amino acids were all characterized. Major epitopes for human IgG(1) to IgG(4) are almost identical. This is the first study to map the sequential epitopes for human IgE and IgG(4) subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis. Copyright © 2011 S. Karger AG, Basel.

Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein.

PubMed

Xinjun, Lv; Xuejun, Ma; Lihua, Wang; Hao, Li; Xinxin, Shen; Pengcheng, Yu; Qing, Tang; Guodong, Liang

2012-05-01

Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus. Copyright © 2012 Elsevier B.V. All rights reserved.

Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies.

PubMed

Godlewska, Marlena; Czarnocka, Barbara; Gora, Monika

2012-09-01

Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders.

A recombinant flagellin fragment, which includes the epitopes flg22 and flgII-28, provides a useful tool to study flagellin-triggered immunity

USDA-ARS?s Scientific Manuscript database

Plants and animals both independently evolved the ability to recognize flagellin (also called FliC), the building block of the bacterial flagellum, as part of their innate immune response. Most plants recognize one or two short epitopes of FliC: flg22 and flgII-28. However, since most research in pl...

Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

PubMed

Sette, Alessandro; Grey, Howard; Oseroff, Carla; Peters, Bjoern; Moutaftsi, Magdalini; Crotty, Shane; Assarsson, Erika; Greenbaum, Jay; Kim, Yohan; Kolla, Ravi; Tscharke, David; Koelle, David; Johnson, R Paul; Blum, Janice; Head, Steven; Sidney, John

2009-12-30

In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

PubMed Central

Müller, Thomas; Dietzschold, Bernhard; Ertl, Hildegund; Fooks, Anthony R.; Freuling, Conrad; Fehlner-Gardiner, Christine; Kliemt, Jeannette; Meslin, Francois X.; Rupprecht, Charles E.; Tordo, Noël; Wanderler, Alexander I.; Kieny, Marie Paule

2009-01-01

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans. PMID:19888334

SdAb heterodimer formation using leucine zippers

NASA Astrophysics Data System (ADS)

Goldman, Ellen R.; Anderson, George P.; Brozozog-Lee, P. Audrey; Zabetakis, Dan

2013-05-01

Single domain antibodies (sdAb) are variable domains cloned from camel, llama, or shark heavy chain only antibodies, and are among the smallest known naturally derived antigen binding fragments. SdAb derived from immunized llamas are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. We hypothesized that the ability to produce heterodimeric sdAb would enable reagents with the robust characteristics of component sdAb, but with dramatically improved overall affinity through increased avidity. Previously we had constructed multimeric sdAb by genetically linking sdAb that bind non-overlapping epitopes on the toxin, ricin. In this work we explored a more flexible approach; the construction of multivalent binding reagents using multimerization domains. We expressed anti-ricin sdAb that recognize different epitopes on the toxin as fusions with differently charged leucine zippers. When the initially produced homodimers are mixed the leucine zipper domains will pair to produce heterodimers. We used fluorescence resonance energy transfer to confirm heterodimer formation. Surface plasmon resonance, circular dichroism, enzyme linked immunosorbent assays, and fluid array assays were used to characterize the multimer constructs, and evaluate their utility in toxin detection.

The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

PubMed Central

Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

2016-01-01

Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

Characterization of Sicilian strains of spotted fever group rickettsiae by using monoclonal antibodies.

PubMed Central

Vitale, G; Di Stefano, R; Damiani, G; Mansueto, S

1989-01-01

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by nine fusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated from a Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect immunofluorescence (IIF) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally in male guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although they produced different titers to the various strains, possibly an indication that they recognized an antigen common to spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patient sera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopes as were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in the expression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses of Boutonneuse fever patients to these epitopes. PMID:2473092

Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies

PubMed Central

Zhai, Yougang; Zhong, Zhenyu; Zariffard, Mohammadreza; Spear, Gregory T.; Qiao, Liang

2013-01-01

Two conserved epitopes, located in the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41, are recognized by two HIV-1 broadly neutralizing antibodies 2F5 and 4E10, and are promising targets for vaccine design in efforts to elicit anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 or 4E10 epitope or the MPER domain into D-E loop of BPV L1 respectively. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting single epitopes. Oral immunization of mice with the chimeric VLPs displaying the 2F5 epitope or MPER domain elicited epitope-specific serum IgGs and mucosal secretory IgAs. The induced antibodies specifically recognized the native conformation of MPER in the context of HIV-1 envelope protein. The antibodies induced by chimeric VLPs presenting MPER domain are able to partially neutralize HIV-1 viruses from clade B and clade C. PMID:24055348

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

PubMed

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

2016-05-05

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

PubMed Central

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

2016-01-01

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

PubMed

Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

2015-01-01

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Oxidation-Specific Epitopes are Danger Associated Molecular Patterns Recognized by Pattern Recognition Receptors of Innate Immunity

PubMed Central

Miller, Yury I.; Choi, Soo-Ho; Wiesner, Philipp; Fang, Longhou; Harkewicz, Richard; Hartvigsen, Karsten; Boullier, Agnès; Gonen, Ayelet; Diehl, Cody J.; Que, Xuchu; Montano, Erica; Shaw, Peter X.; Tsimikas, Sotirios; Binder, Christoph J.; Witztum, Joseph L.

2010-01-01

Oxidation reactions are vital parts of metabolism and signal transduction. However, they also produce reactive oxygen species, which damage lipids, proteins and DNA, generating “oxidation-specific” epitopes. In this review, we will discuss the hypothesis that such common oxidation-specific epitopes are a major target of innate immunity, recognized by a variety of “pattern recognition receptors” (PRRs). By analogy with microbial “pathogen associated molecular patterns” (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent “danger (or damage) associated molecular patterns” (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Further, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation-specific epitopes, such PAMPs provided a strong secondary selecting pressure for the same set of oxidation-specific PRRs as well. Because lipid peroxidation is ubiquitous and a major component of the inflammatory state associated with atherosclerosis, the understanding that oxidation-specific epitopes are DAMPs, and thus the target of multiple arcs of innate immunity, provides novel insights into the pathogenesis of atherosclerosis. As examples, we show that both cellular and soluble PRRs, such as CD36, toll-like receptor-4, natural antibodies, and CRP recognize common oxidation-specific DAMPs, such as oxidized phospholipids and oxidized cholesteryl esters, and mediate a variety of immune responses, from expression of proinflammatory genes to excessive intracellular lipoprotein accumulation to atheroprotective humoral immunity. These insights may lead to improved understanding of inflammation and atherogenesis and suggest new approaches to diagnosis and therapy. PMID:21252151

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

PubMed

Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

2015-02-01

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

PubMed Central

Hjelm, Barbara; Forsström, Björn; Löfblom, John; Rockberg, Johan; Uhlén, Mathias

2012-01-01

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar. PMID:23284606

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

PubMed

Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

2002-10-01

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

PubMed Central

Neophytou, P I; Roep, B O; Arden, S D; Muir, E M; Duinkerken, G; Kallan, A; de Vries, R R; Hutton, J C

1996-01-01

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases. PMID:8700877

Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide with six overlapping epitopes, evolved following polyploidization.

PubMed

Ozuna, Carmen V; Iehisa, Julio C M; Giménez, María J; Alvarez, Juan B; Sousa, Carolina; Barro, Francisco

2015-06-01

The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides. © 2015 Society for Experimental Biology and John Wiley & Sons Ltd.

Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

PubMed Central

Kunwar, Pratima; Hawkins, Natalie; Dinges, Warren L.; Liu, Yi; Gabriel, Erin E.; Swan, David A.; Stevens, Claire E.; Maenza, Janine; Collier, Ann C.; Mullins, James I.; Hertz, Tomer; Yu, Xuesong; Horton, Helen

2013-01-01

A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8+ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8+ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8+ T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8+ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009). Moreover, subjects possessing CD8+ T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021). The association between viral control and the breadth of conserved CD8+ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215). The associations with viral control were independent of functional avidity of CD8+ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8+ T cell responses to multiple conserved epitopes of HIV-1. PMID:23741326

Regions of recognition by blocking antibodies on the light chain of botulinum neurotoxin A: antigenic structure of the entire toxin.

PubMed

Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair

2011-06-01

The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1-453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed. Copyright © 2010 Elsevier GmbH. All rights reserved.

Similar Responses of Intestinal T Cells From Untreated Children and Adults With Celiac Disease to Deamidated Gluten Epitopes.

PubMed

Ráki, Melinda; Dahal-Koirala, Shiva; Yu, Hao; Korponay-Szabó, Ilma R; Gyimesi, Judit; Castillejo, Gemma; Jahnsen, Jørgen; Qiao, Shuo-Wang; Sollid, Ludvig M

2017-09-01

Celiac disease is a chronic small intestinal inflammatory disorder mediated by an immune response to gluten peptides in genetically susceptible individuals. Celiac disease is often diagnosed in early childhood, but some patients receive a diagnosis late in life. It is uncertain whether pediatric celiac disease is distinct from adult celiac disease. It has been proposed that gluten-reactive T cells in children recognize deamidated and native gluten epitopes, whereas T cells from adults only recognize deamidated gluten peptides. We studied the repertoire of gluten epitopes recognized by T cells from children and adults. We examined T-cell responses against gluten by generating T-cell lines and T-cell clones from intestinal biopsies of adults and children and tested proliferative response to various gluten peptides. We analyzed T cells from 14 children (2-5 years old) at high risk for celiac disease who were followed for celiac disease development. We also analyzed T cells from 6 adults (26-55 years old) with untreated celiac disease. All children and adults were positive for HLA-DQ2.5. Biopsies were incubated with gluten digested with chymotrypsin (modified or unmodified by the enzyme transglutaminase 2) or the peptic-tryptic digest of gliadin (in native and deamidated forms) before T-cell collection. Levels of T-cell responses were higher to deamidated gluten than to native gluten in children and adults. T cells from children and adults each reacted to multiple gluten epitopes. Several T-cell clones were cross-reactive, especially clones that recognized epitopes from γ-and ω-gliadin. About half of the generated T-cell clones from children and adults reacted to unknown epitopes. T-cell responses to different gluten peptides appear to be similar between adults and children at the time of diagnosis of celiac disease. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another

PubMed Central

Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

2017-01-01

Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another’s binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen’s unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties. PMID:28060885

Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another.

PubMed

Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

2017-01-01

Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another's binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen's unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.

Depigmented allergoids reveal new epitopes with capacity to induce IgG blocking antibodies.

PubMed

López-Matas, M Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

2013-01-01

The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.

Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes.

PubMed

Bateman, E A L; Ardern-Jones, M R; Ogg, G S

2008-11-01

Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. To identify commonly recognized CD4(+) T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA-restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 ELISpots. Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA-DQB1(*)06 and -DPB1(*)0401 were characterized in detail. Significantly higher ex vivo IL-4 and lower IFN-gamma responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL-10 responses were significantly lower in those with AD in the individuals with HLA-DPB1(*)0401. We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4(+) T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.

Meta-analysis of immune epitope data for all Plasmodia: overview and applications for malarial immunobiology and vaccine-related issues

PubMed Central

Vaughan, K.; Blythe, M.; Greenbaum, J.; Zhang, Q.; Peters, B.; Doolan, D. L.; Sette, A.

2012-01-01

Summary We present a comprehensive meta-analysis of more than 500 references, describing nearly 5000 unique B cell and T cell epitopes derived from the Plasmodium genus, and detailing thousands of immunological assays. This is the first inventory of epitope data related to malaria-specific immunology, plasmodial pathogenesis, and vaccine performance. The survey included host and pathogen species distribution of epitopes, the number of antibody vs. CD4+ and CD8+ T cell epitopes, the genomic distribution of recognized epitopes, variance among epitopes from different parasite strains, and the characterization of protective epitopes and of epitopes associated with parasite evasion of the host immune response. The results identify knowledge gaps and areas for further investigation. This information has relevance to issues, such as the identification of epitopes and antigens associated with protective immunity, the design and development of candidate malaria vaccines, and characterization of immune response to strain polymorphisms. PMID:19149776

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

PubMed Central

Lee, Mey-Fann; Chang, Chia-Wei; Song, Pei-Pong; Hwang, Guang-Yuh; Lin, Shyh-Jye

2015-01-01

Purpose Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. Methods The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. Results Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. Conclusions Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy. PMID:25749772

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2.

PubMed

Lee, Mey Fann; Chang, Chia Wei; Song, Pei Pong; Hwang, Guang Yuh; Lin, Shyh Jye; Chen, Yi Hsing

2015-07-01

Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

PubMed

Carboni, Filippo; Adamo, Roberto; Fabbrini, Monica; De Ricco, Riccardo; Cattaneo, Vittorio; Brogioni, Barbara; Veggi, Daniele; Pinto, Vittoria; Passalacqua, Irene; Oldrini, Davide; Rappuoli, Rino; Malito, Enrico; Margarit, Immaculada Y Ros; Berti, Francesco

2017-05-09

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

PubMed Central

Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

2016-01-01

ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines. PMID:26819303

Mismatch in epitope specificities between IFNγ inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.

PubMed

Woods, Katherine; Knights, Ashley J; Anaka, Matthew; Schittenhelm, Ralf B; Purcell, Anthony W; Behren, Andreas; Cebon, Jonathan

2016-01-01

A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes. We assessed steady state and IFNγ-induced immunoproteasome expression in melanoma cells. Using epitope specific T-lymphocyte clones, we studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Our experimental model allowed comparison of the processing of three distinct epitopes from a single antigen presented on the same HLA complex. We further investigated processing of these epitopes by direct inhibition, or siRNA mediated knockdown, of the immunoproteasome catalytic subunit LMP7. Our data demonstrated a profound difference in the way in which immunogenic T-lymphocyte epitopes are presented by melanoma cells under IFNγ inflammatory versus non-inflammatory conditions. These alterations led to significant changes in the ability of T-lymphocytes to recognize and target melanoma cells. Our results illustrate a little-studied mechanism of immune escape by tumor cells which, with appropriate understanding and treatment, may be reversible. These data have implications for the design of cancer vaccines and adoptive T cell therapies.

Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.

PubMed

Prickett, S R; Voskamp, A L; Phan, T; Dacumos-Hill, A; Mannering, S I; Rolland, J M; O'Hehir, R E

2013-06-01

Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (≤ 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays. Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations. © 2013 John Wiley & Sons Ltd.

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

DOE PAGES

Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

2012-12-13

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

Identification and characterization of haemagglutinin epitopes of Avibacterium paragallinarum serovar C.

PubMed

Noro, Taichi; Oishi, Eiji; Kaneshige, Takahiro; Yaguchi, Kazuhiko; Amimoto, Katsuhiko; Shimizu, Mitsugu

2008-10-15

The objectives of this study were to identify haemagglutinin (HA) epitopes of Avibacterium paragallinarum serovar C that are capable of eliciting haemagglutination inhibition (HI) antibody, and to investigate their immunogenic role. Three conformational epitopes were detected on HA by blocking ELISA and immuno-dot blot analysis using a panel of five monoclonal antibodies (MAbs) with HI activity, designated 8C1C, 4G8B, 24E4D, 11E11B, and 10D1A. The minimum DNA regions coding these three epitopes were 3195, 2862, and 807bp in size, and mapped within a gene with 6117bp. Nine DNA fragments of various lengths were prepared, and their recombinant proteins were generated in E. coli. One recombinant protein, designated HPC5.5, was recognized by MAb 8C1C, and had strong ability to adsorb HI antibody to Av. paragallinarum serovar C. Other recombinant proteins designated HPC5.1, HPC4.8, and HPC2.5 did not react with MAb 8C1C and only slightly adsorbed HI antibody. All chickens immunized once with HPC5.5 did not show any typical clinical signs such as nasal discharge or facial edema against challenge inoculation with Av. paragallinarum serovar C. However, HPC5.1, which was recognized by four MAbs (not including MAb 8C1C), showed only partial protective immunity in five of eight immunized chickens. The results suggest that the HA epitope recognized by MAb 8C1C is the major epitope responsible for eliciting HI antibody, and HPC5.5 is a practical candidate protein to develop a new vaccine against avian infectious coryza caused by Av. paragallinarum serovar C.

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

PubMed Central

Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

2015-01-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

PubMed

Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

2015-05-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

PubMed Central

Aw-Yong, Kam Leng; Sam, I-Ching; Koh, Mia Tuang

2016-01-01

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. PMID:27806091

The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes

PubMed Central

Dørum, Siri; Arntzen, Magnus Ø.; Qiao, Shuo-Wang; Holm, Anders; Koehler, Christian J.; Thiede, Bernd; Sollid, Ludvig M.; Fleckenstein, Burkhard

2010-01-01

Background Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. Methods A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. Results We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. Conclusion TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease. PMID:21124911

Identification of a conformational neutralizing epitope on the VP1 protein of type A foot-and-mouth disease virus.

PubMed

Liu, Wenming; Yang, Baolin; Wang, Mingxia; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype A and protect against future pandemics. In this study, we generated a serotype A FMDV-specific potent neutralizing monoclonal antibody (MAb), 6C9, which recognizes a conformation-dependent epitope. MAb 6C9 potently neutralized FMDV A/XJBC/CHA/2010 with a 50% neutralization titer (NT 50 ) of 4096. Screening of a phage-displayed random 12-mer peptide library revealed that MAb 6C9 bound to phages displaying the consensus motif YxxPxGDLG, which is highly homologous to the 135 YxxPxxxxxGDLG 147 motif found in the serotype A FMDV virus-encoded structural protein VP1. To further verify the authentic epitope recognized by MAb 6C9, two FMDV A/XJBC/CHA/2010 mutant viruses, P138A and G144A, were generated using a reverse genetic system. Subsequent micro-neutralization assays and double-antibody sandwich (DAS) ELISA analyses revealed that the Pro 138 and Gly 144 residues of the conformational epitope that are recognized by 6C9 are important for MAb 6C9 binding. Importantly, the epitope 135 YxxPxxxxxGDLG 147 was highly conserved among different topotypes of serotype A FMDV strains in a sequence alignment analysis. Thus, the results of this study could have potential applications in the development of novel epitope-based vaccines and suitable a MAb-based diagnostic method for the detection of serotype A FMDV and the quantitation of antibodies against this serotype. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model*

PubMed Central

Moayeri, Mahtab; Leysath, Clinton E.; Tremblay, Jacqueline M.; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H.; Shoemaker, Charles B.

2015-01-01

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from “pre-pore” to its SDS and heat-resistant “pore” conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. PMID:25564615

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus

PubMed Central

Azzouz, Doua F.; Martin, Gabriel V.; Arnoux, Fanny; Balandraud, Nathalie; Martin, Thierry; Dubucquoi, Sylvain; Hachulla, Eric; Farge-Bancel, Dominique; Tiev, Kiet; Cabane, Jean; Bardin, Nathalie; Chiche, Laurent; Martin, Marielle; Caillet, Eléonore C.; Kanaan, Sami B.; Harlé, Jean Robert; Granel, Brigitte; Diot, Elisabeth; Roudier, Jean; Auger, Isabelle; Lambert, Nathalie C.

2016-01-01

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10−4) and 60% versus 28% (P = 3.10−8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10−10) and 82% (P = 5.10−13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3’-5’ exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis. PMID:27617966

A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

PubMed

Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

2015-03-06

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of CTL Recognized Epitopes on Human Breast Tumors

DTIC Science & Technology

1996-09-01

maturation and effector function of cellular immune cytotoxic effectors such as CTL (11). (c) The epitopes defined on tumor Ag are self-peptides of...have been reported to be expressed in breast and ovarian cancer cells (18), and they apparently function by maintaining the undifferentiated state...Body of the Report The purpose of the present work continues to be the characterization of the functional significance of the CTL epitopes as potential

Breast Mucin Tumor-Specific Epitopes for Cancer Immunotherapy

DTIC Science & Technology

1998-09-01

reactivity with tumor-specific monoclonal antibodies show that antigenicity is maximized with the 40 amino acid MUC1-mtr2. By contrast, the MUC1-mtr3...associated mucins (7). The presence of tumor-specific epitopes is evidenced by the development of many monoclonal antibodies (mAb) that recognize...P1-P5 in the tandem repeat sequence (7). This epitope was identified by competition of antibody binding to tumor- specific mucin by synthetic

Analysis of Individuals from a Dengue-Endemic Region Helps Define the Footprint and Repertoire of Antibodies Targeting Dengue Virus 3 Type-Specific Epitopes.

PubMed

Andrade, Daniela V; Katzelnick, Leah C; Widman, Doug G; Balmaseda, Angel; de Silva, Aravinda M; Baric, Ralph S; Harris, Eva

2017-09-19

The four dengue virus serotypes (DENV1 to 4) cause dengue, a major public health problem worldwide. Individuals exposed to primary DENV infections develop serotype-specific neutralizing antibodies, including strongly neutralizing antibodies targeting quaternary epitopes. To date, no studies have measured the levels and kinetics of serum antibodies directed to such epitopes among populations in regions where dengue is endemic. Here, we use a recombinant DENV4 (rDENV4/3-M14) displaying a major DENV3 type-specific quaternary epitope recognized by human monoclonal antibody 5J7 to measure the proportion, magnitude, and kinetics of DENV3 type-specific neutralizing antibody responses targeting this epitope. Primary DENV3 sera from 30 individuals in a dengue hospital-based study in Nicaragua were studied 3, 6, 12, and 18 months post-infection, alongside samples collected annually 1 to 4 years post-primary DENV3 infection from 10 individuals in a cohort study in Nicaragua. We found substantial individual variation in the proportion of DENV3 type-specific neutralizing antibody titers attributed to the 5J7 epitope (range, 0 to 100%), with the mean significantly increasing from 22.6% to 41.4% from 3 to 18 months. We extended the transplanted DENV3 5J7 epitope on the virion (rDENV4/3-M16), resulting in increased recognition in several individuals, helping define the footprint of the epitope. However, 37% and 13% of the subjects still showed little to no recognition of the 5J7 epitope at 3 and 18 months, respectively, indicating that one or more additional DENV3 type-specific epitopes exist. Overall, this study demonstrates how DENV-immune plasma from populations from areas of endemicity, when coupled with structurally guided recombinant viruses, can help characterize the epitope-specific neutralizing antibody response in natural DENV infections, with direct implications for design and evaluation of dengue vaccines. IMPORTANCE The four serotypes of dengue virus cause dengue, a major public health burden worldwide, yet it has been challenging to develop a vaccine that is safe and equally effective against all four serotypes. More in-depth characterization of natural human neutralizing antibody responses is needed to identify determinants of protective antibody responses to all DENV serotypes. Here, we use hospital and cohort studies in a region where dengue is endemic to assess the proportion and kinetics of the DENV3 neutralizing antibody response directed to a quaternary epitope on DENV3 recognized by strongly neutralizing human monoclonal antibody 5J7, which was transplanted into a DENV4 backbone. We show that many individuals recognized the 5J7 epitope, but to various degrees over time, suggesting that additional DENV3-specific epitopes likely exist. Thus, characterization of epitope-specific neutralizing antibody responses in natural DENV infections can help define the footprint and repertoire of antibodies directed to DENV3 type-specific epitopes, with implications for dengue vaccine development. Copyright © 2017 Andrade et al.

Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

PubMed Central

Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

2015-01-01

Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

Characterization of CD8+ T-cell response in acute and resolved hepatitis A virus infection.

PubMed

Schulte, I; Hitziger, T; Giugliano, S; Timm, J; Gold, H; Heinemann, F M; Khudyakov, Y; Strasser, M; König, C; Castermans, E; Mok, J Y; van Esch, W J E; Bertoletti, A; Schumacher, T N; Roggendorf, M

2011-02-01

In contrast to the infection with other hepatotropic viruses, hepatitis A virus (HAV) always causes acute self-limited hepatitis, although the role for virus-specific CD8 T cells in viral containment is unclear. Herein, we analyzed the T cell response in patients with acute hepatitis by utilizing a set of overlapping peptides and predicted HLA-A2 binders from the polyprotein. A set of 11 predicted peptides from the HAV polyprotein, identified as potential binders, were synthesized. Peripheral blood mononuclear cells (PBMCs) from patients were tested for IFNγ secretion after stimulation with these peptides and ex vivo with HLA-A2 tetramers. Phenotyping was carried out by staining with the activation marker CD38 and the memory marker CD127. Eight out of 11 predicted HLA-A2 binders showed a high binding affinity and five of them were recognized by CD8+ T cells from patients with hepatitis A. There were significant differences in the magnitude of the responses to these five peptides. One was reproducibly immunodominant and the only one detectable ex vivo by tetramer staining of CD8+ T cells. These cells have an activated phenotype (CD38hi CD127lo) during acute infection. Three additional epitopes were identified in HLA-A2 negative patients, most likely representing epitopes restricted by other HLA-class I-alleles (HLA-A11, B35, B40). Patients with acute hepatitis A have a strong multi-specific T cell response detected by ICS. With the tetramer carrying the dominant HLA-A2 epitope, HAV-specific and activated CD8+ T cells could be detected ex vivo. This first description of the HAV specific CTL-epitopes will allow future studies on strength, breadth, and kinetics of the T-cell response in hepatitis A. Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

PubMed Central

Chen, W; Qin, H; Chesebro, B; Cheever, M A

1996-01-01

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV. PMID:8892898

Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

PubMed Central

López-Matas, M. Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

2013-01-01

Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT. PMID:24222901

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

PubMed

Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P; Takeda, Makoto

2016-08-02

Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.

Identification of two novel immunodominant UreB CD4(+) T cell epitopes in Helicobacter pylori infected subjects.

PubMed

Yang, Wu-Chen; Chen, Li; Li, Hai-Bo; Li, Bin; Hu, Jian; Zhang, Jin-Yong; Yang, Shi-Ming; Zou, Quan-Ming; Guo, Hong; Wu, Chao

2013-02-06

An epitope-based vaccine is a promising option for treating Helicobacter pylori (H. pylori) infection. Epitope mapping is the first step in designing an epitope-based vaccine. A pivotal role of CD4(+) T cells in protection against H. pylori has been accepted, but few Th epitopes have been identified. In this study, two novel UreB CD4(+) T cell epitopes were identified using PBMCs obtained from two H. pylori infected subjects. We determined the restriction molecules by antibody blocking and used various Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs) with different HLA alleles as APCs to present peptides to CD4(+) T cells. These epitopes were DRB1*1404-restricted UreB(373-385) and DRB1*0803-restricted UreB(438-452). The T cells specific to these epitopes not only recognized autologous DCs loaded with recombinant UreB but also those pulsed with H. pylori whole cell lysates, suggesting that these epitope peptides are naturally processed. These epitopes have important value for designing an effective H. pylori vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

Specificities of Human CD4+ T Cell Responses to an Inactivated Flavivirus Vaccine and Infection: Correlation with Structure and Epitope Prediction

PubMed Central

Schwaiger, Julia; Aberle, Judith H.; Stiasny, Karin; Knapp, Bernhard; Schreiner, Wolfgang; Fae, Ingrid; Fischer, Gottfried; Scheinost, Ondrej; Chmelik, Vaclav

2014-01-01

ABSTRACT Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4+ T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4+ T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4+ T cell epitopes. IMPORTANCE Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection. PMID:24789782

Identification and characterization of epitopes on Plasmodium knowlesi merozoite surface protein-142 (MSP-142) using synthetic peptide library and phage display library.

PubMed

Cheong, Fei Wen; Fong, Mun Yik; Lau, Yee Ling

2016-02-01

Plasmodium knowlesi can cause potentially life threatening human malaria. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential target for malaria blood stage vaccine, and for diagnosis of malaria. Two epitope mapping techniques were used to identify the potential epitopes within P. knowlesi MSP-142. Nine and 14 potential epitopes were identified using overlapping synthetic peptide library and phage display library, respectively. Two regions on P. knowlesi MSP-142 (amino acid residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two of the prominent epitopes, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were evaluated using mouse model. P10- and P31-immunized mouse sera reacted with recombinant P. knowlesi MSP-142, with the IgG isotype distribution of IgG2b>IgG1>IgG2a>IgG3. Significant higher level of cytokines interferon-gamma and interleukin-2 was detected in P31-immunized mice. Both P10 and P31 could be the suitable epitope candidates to be used in malaria vaccine designs and immunodiagnostic assays, provided further evaluation is needed to validate the potential uses of these epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

Computational and Experimental Validation of B and T-Cell Epitopes of the In Vivo Immune Response to a Novel Malarial Antigen

DTIC Science & Technology

2013-08-16

approach in the context of a novel, immunologically relevant antigen. The limited accuracy of the tested algorithms to predict the in vivo immune responses...overlapping peptides spanning the entire sequence are individually tested for antibody interacting residues. Conformational B cell epitopes, in contrast...a blind assessment of this approach in the context of a novel, immunologically relevant antigen. The limited accuracy of the tested algorithms to

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

PubMed Central

Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

2011-01-01

Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana. PMID:21826225

The epitopes that cause cross-reactions between peanuts and tree nuts

USDA-ARS?s Scientific Manuscript database

Many peanut allergic individuals also have allergies to tree nuts. Our previous work has shown that there are epitopes with different amino acid sequences, but similar physical and chemical properties are recognized by the same IgE molecule. Anti-Ara h 2 monoclonal antibodies were produced. They we...

Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes.

PubMed

Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2015-05-01

Identification and characterization of CD8(+) T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8(+) T cells have been only partially identified. In this study, we sought to identify CD8(+) T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8(+) T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10(-11)) and positively associated with CD4 count (P = 1.2 × 10(-11)), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8(+) T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted and 3 HLA-B*67:01-restricted CTLs, suggesting that these CTLs play a predominant role in HIV-1 control. The 13 CTLs showed synergistic effects on HIV-1 control. Twelve out of these 13 epitopes were recognized as conserved or cross-recognized ones. These findings strongly suggest that these 12 epitopes are candidates for antigens for AIDS vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes

PubMed Central

Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi

2015-01-01

ABSTRACT Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8+ T cells have been only partially identified. In this study, we sought to identify CD8+ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8+ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10−11) and positively associated with CD4 count (P = 1.2 × 10−11), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8+ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. IMPORTANCE HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted and 3 HLA-B*67:01-restricted CTLs, suggesting that these CTLs play a predominant role in HIV-1 control. The 13 CTLs showed synergistic effects on HIV-1 control. Twelve out of these 13 epitopes were recognized as conserved or cross-recognized ones. These findings strongly suggest that these 12 epitopes are candidates for antigens for AIDS vaccines. PMID:25741000

Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity.

PubMed

Zaman, Mehfuz; Chandrudu, Saranya; Giddam, Ashwini K; Reiman, Jennifer; Skwarczynski, Mariusz; McPhun, Virginia; Moyle, Peter M; Batzloff, Michael R; Good, Michael F; Toth, Istvan

2014-12-01

Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

Mapping the B cell epitopes within the major capsid protein L1 of human papillomavirus type 16.

PubMed

Wang, Aiping; Li, Ning; Zhou, Jingming; Chen, Yumei; Jiang, Min; Qi, Yanhua; Liu, Hongliang; Liu, Yankai; Liu, Dongmin; Zhao, Jianguo; Wang, Yanwei; Zhang, Gaiping

2018-06-26

Persistent infection with human papillomavirus type16 (HPV16) has much association with the development of cervical cancer. L1 is the major capsid protein of HPV, it has been well investigated as a potential vaccine candidate. However, B cell epitopes present on L1 have not been well characterized. To identify the potential B-cell antigenic epitopes within HPV16 L1 protein, sixteen serial overlapping truncations (H1-H16) covering the whole region were expressed in E. coli and used in mice immunization. The mice antisera were tested in ELISA binding, IFA and HI assays. Finally, four fragments (H2, H4, H11, H12) were found to contain B cell epitopes of HPV16 L1 protein in ELISA and IFA assays, three fragments (H2, H3, H9) might contain neutralizing epitopes of HPV16 L1 protein in HI assay. Among them, H11 and H12 fragments contain B cell epitopes have never been reported before, and H3 was found as hemagglutination inhibition epitope for the first time. This work provides new insights to B cell epitopes on HPV16 L1 protein. Several new epitopes were identified and may provide some guidance for HPV16 subunit vaccine design. The results of this study might open new perspectives on the antibody-antigen reaction and have important implications for the development of epitopes-based protective HPV16 vaccines. Copyright © 2018. Published by Elsevier B.V.

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule.

PubMed

Atassi, M Zouhair; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger; Dolimbek, Behzod Z

2012-01-01

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.

The Waardenburg Syndrome Type 4 Gene, SOX10, Is a Novel Tumor-associated Antigen Identified in a Patient with a Dramatic Response to Immunotherapy

PubMed Central

Khong, Hung T.; Rosenberg, Steven A.

2008-01-01

In this study, we have identified, for the first time, the presence of de novo cellular immune reactivity against the transcription factor SOX10, using tumor-infiltrating lymphocytes obtained from a patient who experienced a dramatic clinical response to immunotherapy. SOX10 acts as a critical transactivator of tyrosinase-related protein-2 during melanoblast development and a potent transactivator of micropthalmia-associated transcription factor, which is considered to be a master gene that controls the development and postnatal survival of melanocytes. Mutations in SOX10 result in Waardenburg syndrome type 4. The overlapping epitopes AWISKPPGV and SAWISKPPGV, designated SOX10: 332–340 and SOX10: 331–340, respectively, were recognized by tumor-infiltrating lymphocyte clone M37 in an HLA-A2-restricted fashion. PMID:12036907

Structures of MART-1 26/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K

2008-09-17

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning aminomore » acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.« less

Structures of MART-126/27–35 Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

PubMed Central

Borbulevych, Oleg Y.; Insaidoo, Francis K.; Baxter, Tiffany K.; Powell, Daniel J.; Johnson, Laura A.; Restifo, Nicholas P.; Baker, Brian M.

2007-01-01

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26–35 decamer, although only the 27–35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26–35 and 27–35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-126/27–35-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27–35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone. PMID:17719062

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

PubMed

Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

2000-04-01

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

PubMed

Clavarino, Giovanna; Gauthier, Arnaud; Hellmark, Thomas; Carron, Pierre-Louis; Giovannini, Diane; Colliard, Sophie; Dragon-Durey, Marie-Agnès; Segelmark, Mårten; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

2018-04-12

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

PubMed Central

Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

2016-01-01

Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

Variable content and distribution of arabinogalactan proteins in banana (Musa spp.) under low temperature stress.

PubMed

Yan, Yonglian; Takáč, Tomáš; Li, Xiaoquan; Chen, Houbin; Wang, Yingying; Xu, Enfeng; Xie, Ling; Su, Zhaohua; Šamaj, Jozef; Xu, Chunxiang

2015-01-01

Information on the spatial distribution of arabinogalactan proteins (AGPs) in plant organs and tissues during plant reactions to low temperature (LT) is limited. In this study, the extracellular distribution of AGPs in banana leaves and roots, and their changes under LT stress were investigated in two genotypes differing in chilling tolerance, by immuno-techniques using 17 monoclonal antibodies against different AGP epitopes. Changes in total classical AGPs in banana leaves were also tested. The results showed that AGP epitopes recognized by JIM4, JIM14, JIM16, and CCRC-M32 antibodies were primarily distributed in leaf veins, while those recognized by JIM8, JIM13, JIM15, and PN16.4B4 antibodies exhibited predominant sclerenchymal localization. Epitopes recognized by LM2, LM14, and MAC207 antibodies were distributed in both epidermal and mesophyll cells. Both genotypes accumulated classical AGPs in leaves under LT treatment, and the chilling tolerant genotype contained higher classical AGPs at each temperature treatment. The abundance of JIM4 and JIM16 epitopes in the chilling-sensitive genotype decreased slightly after LT treatment, and this trend was opposite for the tolerant one. LT induced accumulation of LM2- and LM14-immunoreactive AGPs in the tolerant genotype compared to the sensitive one, especially in phloem and mesophyll cells. These epitopes thus might play important roles in banana LT tolerance. Different AGP components also showed differential distribution patterns in banana roots. In general, banana roots started to accumulate AGPs under LT treatment earlier than leaves. The levels of AGPs recognized by MAC207 and JIM13 antibodies in the control roots of the tolerant genotype were higher than in the chilling sensitive one. Furthermore, the chilling tolerant genotype showed high immuno-reactivity against JIM13 antibody. These results indicate that several AGPs are likely involved in banana tolerance to chilling injury.

Variable content and distribution of arabinogalactan proteins in banana (Musa spp.) under low temperature stress

PubMed Central

Yan, Yonglian; Takáč, Tomáš; Li, Xiaoquan; Chen, Houbin; Wang, Yingying; Xu, Enfeng; Xie, Ling; Su, Zhaohua; Šamaj, Jozef; Xu, Chunxiang

2015-01-01

Information on the spatial distribution of arabinogalactan proteins (AGPs) in plant organs and tissues during plant reactions to low temperature (LT) is limited. In this study, the extracellular distribution of AGPs in banana leaves and roots, and their changes under LT stress were investigated in two genotypes differing in chilling tolerance, by immuno-techniques using 17 monoclonal antibodies against different AGP epitopes. Changes in total classical AGPs in banana leaves were also tested. The results showed that AGP epitopes recognized by JIM4, JIM14, JIM16, and CCRC-M32 antibodies were primarily distributed in leaf veins, while those recognized by JIM8, JIM13, JIM15, and PN16.4B4 antibodies exhibited predominant sclerenchymal localization. Epitopes recognized by LM2, LM14, and MAC207 antibodies were distributed in both epidermal and mesophyll cells. Both genotypes accumulated classical AGPs in leaves under LT treatment, and the chilling tolerant genotype contained higher classical AGPs at each temperature treatment. The abundance of JIM4 and JIM16 epitopes in the chilling-sensitive genotype decreased slightly after LT treatment, and this trend was opposite for the tolerant one. LT induced accumulation of LM2- and LM14-immunoreactive AGPs in the tolerant genotype compared to the sensitive one, especially in phloem and mesophyll cells. These epitopes thus might play important roles in banana LT tolerance. Different AGP components also showed differential distribution patterns in banana roots. In general, banana roots started to accumulate AGPs under LT treatment earlier than leaves. The levels of AGPs recognized by MAC207 and JIM13 antibodies in the control roots of the tolerant genotype were higher than in the chilling sensitive one. Furthermore, the chilling tolerant genotype showed high immuno-reactivity against JIM13 antibody. These results indicate that several AGPs are likely involved in banana tolerance to chilling injury. PMID:26074928

Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury1

PubMed Central

Kulik, Liudmila; Fleming, Sherry D.; Moratz, Chantal; Reuter, Jason W.; Novikov, Aleksey; Chen, Kuan; Andrews, Kathy A.; Markaryan, Adam; Quigg, Richard J.; Silverman, Gregg J.; Tsokos, George C.; Holers, V. Michael

2010-01-01

Intestinal ischemia-reperfusion (IR)3 injury is initiated when natural IgM antibodies recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild type C57BL/6 mice. We identified a novel IgM monoclonal antibody (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. Monoclonal Ab B4 was found to specifically recognize mouse annexin IV. Pre-injection of recombinant annexin IV blocked IR injury in wild type C57BL/6 mice, demonstrating the requirement for recognition of this protein in order to develop IR injury in the context of a complex natural antibody repertoire. Humans were also found to exhibit IgM natural antibodies that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target antigen that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury. PMID:19380783

BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes

PubMed Central

Jespersen, Martin Closter; Peters, Bjoern

2017-01-01

Abstract Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex task. Despite substantial effort, limited advancement has been achieved over the last decade in the accuracy of epitope prediction methods, especially for those that rely on the sequence of the antigen only. Here, we present BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/), a web server for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from solved 3D structures, and on a large collection of linear epitopes downloaded from the IEDB database. The method displays results in a user-friendly and informative way, both for computer-savvy and non-expert users. We believe that BepiPred-2.0 will be a valuable tool for the bioinformatics and immunology community. PMID:28472356

HIV-1 Vaccines Based on Antibody Identification, B Cell Ontogeny, and Epitope Structure.

PubMed

Kwong, Peter D; Mascola, John R

2018-05-15

HIV-1 vaccine development has been stymied by an inability to induce broadly reactive neutralizing antibodies to the envelope (Env) trimer, the sole viral antigen on the virion surface. Antibodies isolated from HIV-1-infected donors, however, have been shown to recognize all major exposed regions of the prefusion-closed Env trimer, and an emerging understanding of the immunological and structural characteristics of these antibodies and the epitopes they recognize is enabling new approaches to vaccine design. Antibody lineage-based design creates immunogens that activate the naive ancestor-B cell of a target antibody lineage and that mature intermediate-B cells toward effective neutralization, with proof of principle achieved with select HIV-1-neutralizing antibody lineages in human-gene knock-in mouse models. Epitope-based vaccine design involves the engineering of sites of Env vulnerability as defined by the recognition of broadly neutralizing antibodies, with cross-reactive neutralizing antibodies elicited in animal models. Both epitope-based and antibody lineage-based HIV-1 vaccine approaches are being readied for human clinical trials. Published by Elsevier Inc.

Epitope mapping of commercial antibodies that detect myocilin.

PubMed

Patterson-Orazem, Athéna C; Hill, Shannon E; Fautsch, Michael P; Lieberman, Raquel L

2018-05-09

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Diverse cross-reactive potential and Vbeta gene usage of an epitope-specific cytotoxic T-lymphocyte population in monkeys immunized with diverse human immunodeficiency virus type 1 Env immunogens.

PubMed

Hulot, Sandrine L; Seaman, Michael S; Sen, Pritha; Autissier, Patrick A; Manuel, Edwin R; Letvin, Norman L

2009-10-01

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vbeta repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8(+) T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8(+) T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vbeta gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

PubMed

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

PubMed Central

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

Identification and characterization of nematode specific protective epitopes of Brugia malayi TRX towards development of synthetic vaccine construct for lymphatic filariasis.

PubMed

Madhumathi, Jayaprakasam; Prince, Prabhu Rajaiah; Anugraha, Gandhirajan; Kiran, Pote; Rao, Donthamsetty Nageswara; Reddy, Maryada Venkata Rami; Kaliraj, Perumal

2010-07-12

Although multi-epitope vaccines have been evaluated for various diseases, they have not yet been investigated for lymphatic filariasis. Here, we report for the first time identification of two immunodominant B epitopes (TRXP1 and TRXP2) from the antioxidant Brugia malayi thioredoxin by studying their immune responses in mice model and human subjects. TRXP1 was also found to harbor a T epitope recognized by human PBMCs and mice splenocytes. Further, the epitopic peptides were synthesized as a single peptide conjugate (PC1) and their prophylactic efficacy was tested in a murine model of filariasis with L3 larvae. PC1 conferred a significantly high protection (75.14%) (P < 0.0001) compared to control (3.7%) and recombinant TRX (63.03%) (P < 0.018) in experimental filariasis. Our results suggest that multi-epitope vaccines could be a promising strategy in the control of lymphatic filariasis.

Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis

PubMed Central

1995-01-01

Lewis rats are susceptible to several forms of experimental arthritis- induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II- restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell- mediated regulation of inflammation. PMID:7869052

HIV-1 epitopes presented by MHC class I types associated with superior immune containment of viremia have highly constrained fitness landscapes.

PubMed

Gorin, Aleksandr M; Du, Yushen; Liu, Franklin Y; Zhang, Tian-Hao; Ng, Hwee L; Hofmann, Christian; Cumberland, William G; Sun, Ren; Yang, Otto O

2017-08-01

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.

Broad cross-reactive T cell receptor repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

PubMed Central

Clute, Shalyn C.; Naumov, Yuri N.; Watkin, Levi B.; Aslan, Nuray; Sullivan, John L.; Thorley-Lawson, David A.; Luzuriaga, Katherine; Welsh, Raymond M.; Puzone, Roberto; Celada, Franco; Selin, Liisa K.

2013-01-01

Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show here, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M158-66 epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1280-288 epitope (GLCTLVAML), that under some conditions heterologous immunity can lead to a significant broadening rather than a narrowing of the T cell receptor repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad rather than narrow T cell repertoire if there is a lack of dominant high affinity clones, and this hypothesis is supported by computer simulation. PMID:21048112

Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

PubMed

Ofran, Yanay; Schlessinger, Avner; Rost, Burkhard

2008-11-01

Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

PubMed Central

2010-01-01

Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong purifying selection acting at the nucleotide level of the associated epitopes indicates that these regions are functionally critical, although the exact reasons behind such sequence conservation remain to be elucidated. PMID:20696039

Structures of protective antibodies reveal sites of vulnerability on Ebola virus.

PubMed

Murin, Charles D; Fusco, Marnie L; Bornholdt, Zachary A; Qiu, Xiangguo; Olinger, Gene G; Zeitlin, Larry; Kobinger, Gary P; Ward, Andrew B; Saphire, Erica Ollmann

2014-12-02

Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.

Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes

PubMed Central

Hasan, Noor Haliza; Ignjatovic, Jagoda; Tarigan, Simson; Peaston, Anne; Hemmatzadeh, Farhid

2016-01-01

A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development. PMID:27362795

Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

PubMed Central

da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M.; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S.

2017-01-01

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. PMID:28081174

Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses.

PubMed

da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S

2017-01-01

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.

Analysis of B-cell epitopes from the allergen Hev b 6.02 revealed by using blocking antibodies.

PubMed

Pedraza-Escalona, Martha; Becerril-Luján, Baltazar; Agundis, Concepción; Domínguez-Ramírez, Lenin; Pereyra, Ali; Riaño-Umbarila, Lidia; Rodríguez-Romero, Adela

2009-02-01

Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.

Alterations in expression of Cat-315 epitope of perineuronal nets during normal ageing, and its modulation by an open-channel NMDA receptor blocker, memantine.

PubMed

Yamada, Jun; Ohgomori, Tomohiro; Jinno, Shozo

2017-06-15

The perineuronal net (PNN), a specialized aggregate of the extracellular matrix, is involved in neuroprotection against oxidative stress, which is now recognized as a major contributor to age-related decline in brain functions. In this study, we investigated the age-related molecular changes of PNNs using monoclonal antibody Cat-315, which recognizes human natural killer-1 (HNK-1) glycan on aggrecan-based PNNs. Western blot analysis showed that the expression levels of Cat-315 epitope in the hippocampus were higher in middle-aged (MA, 12-month-old) mice than in young adult (YA, 2-month-old) mice. Although there were no differences in the expression levels of Cat-315 epitope between old age (OA, 20-month-old) and MA mice, Cat-315 immunoreactivity was also detected in astrocytes of OA mice. To focus on Cat-315 epitope in PNNs, we used YA and MA mice in the following experiments. Optical disector analysis showed that there were no differences in the numbers of Cat-315-positive (Cat-315 + ) PNNs between YA and MA mice. Fluorescence intensity analysis indicated that Cat-315 immunoreactivity in PNNs increased with age in the dorsal hippocampus, which is mainly involved in cognitive functions. Administration of an open-channel blocker of NMDA receptor, memantine, reduced the expression levels of Cat-315 epitope in the hippocampus. Furthermore, the numbers of glutamatergic and GABAergic terminals colocalized with Cat-315 epitope around parvalbumin-positive neurons were decreased by memantine. These findings provide novel insight into the involvement of PNNs in normal brain ageing, and suggest that memantine may counteract the age-related alterations in expression levels of Cat-315 epitope via regulation of its subcellular localization. © 2017 Wiley Periodicals, Inc.

Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice

DTIC Science & Technology

2017-04-06

recognized by mAb 6D8 when assayed by immunofluourescent antibody staining of transfected cells (Figure 5A). As expected, ELISA of cell culture... ELISA using whole EBOV antigen. Mep1 elicited no detectable antibody response after two vaccinations and only a low response after three...vaccinations. Mep2 elicited a detectable response after two vaccinations with a rise in ELISA antibody titer after three vaccinations (Figure 5C

Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

PubMed Central

Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

2015-01-01

The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

Characterization of cross-clade monoclonal antibodies against H5N1 highly pathogenic avian influenza virus and their application to the antigenic analysis of diverse H5 subtype viruses.

PubMed

Gronsang, Dulyatad; Bui, Anh N; Trinh, Dai Q; Bui, Vuong N; Nguyen, Khong V; Can, Minh X; Omatsu, Tsutomu; Mizutani, Tetsuya; Nagai, Makoto; Katayama, Yukie; Thampaisarn, Rapeewan; Ogawa, Haruko; Imai, Kunitoshi

2017-08-01

H5N1 highly pathogenic avian influenza viruses (HPAIVs) are a threat to both animal and public health and require specific and rapid detection for prompt disease control. We produced three neutralizing anti-hemagglutinin (HA) monoclonal antibodies (mAbs) using two clades (2.2 and 2.5) of the H5N1 HPAIV isolated in Japan. Blocking immunofluorescence tests showed that each mAb recognized different epitopes; 3B5.1 and 3B5.2 mAbs against the clade 2.5 virus showed cross-clade reactivity to all 26 strains from clades 1, 2.2, 2.3.2.1, 2.3.2.1a, b, c and 2.3.4, suggesting that the epitope(s) recognized are conserved. Conversely, the 1G5 mAb against the clade 2.2 virus showed reactivity to only clades 1, 2.3.4 and 2.5 strains. An analysis of escape mutants, and some clades of the H5N1 viruses recognized by 3B5.1 and 3B5.2 mAbs, suggested that the mAbs bind to an epitope, including amino acid residues at position 162 in the HA1 protein (R162 and K162). Unexpectedly, however, when five Eurasian-origin H5 low-pathogenic AIV (LPAIV) strains with R162 were examined (EA-nonGsGD clade) as well as two American-origin strains (Am-nonGsGD clade), the mAb recognized only EA-nonGsGD clade strains. The R162 and K162 residues in the HA1 protein were highly conserved among 36 of the 43 H5N1 clades reported, including clades 2.3.2.1a and 2.3.2.1c that are currently circulating in Asia, Africa and Europe. The amino acid residues (158-PTIKRSYNNTNQE-170) in the HA1 protein are probably an epitope responsible for the cross-clade reactivity of the mAbs, considering the epitopes reported elsewhere. The 3B5.1 and 3B5.2 mAbs may be useful for the specific detection of H5N1 HPAIVs circulating in the field.

BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes.

PubMed

Jespersen, Martin Closter; Peters, Bjoern; Nielsen, Morten; Marcatili, Paolo

2017-07-03

Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex task. Despite substantial effort, limited advancement has been achieved over the last decade in the accuracy of epitope prediction methods, especially for those that rely on the sequence of the antigen only. Here, we present BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/), a web server for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from solved 3D structures, and on a large collection of linear epitopes downloaded from the IEDB database. The method displays results in a user-friendly and informative way, both for computer-savvy and non-expert users. We believe that BepiPred-2.0 will be a valuable tool for the bioinformatics and immunology community. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Processing of two latent membrane protein 1 MHC class I epitopes requires tripeptidyl peptidase II involvement.

PubMed

Diekmann, Jan; Adamopoulou, Eleni; Beck, Olaf; Rauser, Georg; Lurati, Sarah; Tenzer, Stefan; Einsele, Hermann; Rammensee, Hans-Georg; Schild, Hansjörg; Topp, Max S

2009-08-01

The EBV Ag latent membrane protein 1 (LMP1) has been described as a potential target for T cell immunotherapy in EBV-related malignancies. However, only a few CD8(+) T cell epitopes are known, and the benefit of LMP1-specific T cell immunotherapy has not yet been proven. In this work, we studied the processing of the two LMP1 HLA-A02-restricted epitopes, YLLEMLRWL and YLQQNWWTL. We found that target cells endogenously expressing the native LMP1 are not recognized by CTLs specific for these epitopes because the N-terminal part of LMP1 limits the efficiency of epitope generation. We further observed that the proteasome is not required for the generation of both epitopes and that the YLLEMLRWL epitope seems to be destroyed by the proteasome, because blocking of proteasomal activities enhanced specific CTL activation. Activation of LMP1-specific CTLs could be significantly reduced after inhibition of the tripeptidyl peptidase II, suggesting a role for this peptidase in the processing of both epitopes. Taken together, our results demonstrate that the MHC class I-restricted LMP1 epitopes studied in this work are two of very few epitopes known to date to be processed proteasome independently by tripeptidyl peptidase II.

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stainmore » electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design.« less

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

PubMed Central

Elliott, Debra H.; Rouelle, Julie; Smira, Ashley; Ndabambi, Nonkululeko; Druz, Aliaksandr; Williamson, Carolyn

2017-01-01

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design. PMID:28076415

Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic.

PubMed

Prickett, Sara R; Voskamp, Astrid L; Dacumos-Hill, April; Symons, Karen; Rolland, Jennifer M; O'Hehir, Robyn E

2011-03-01

Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

PubMed Central

Austin, S. Kyle; Dowd, Kimberly A.; Shrestha, Bimmi; Nelson, Christopher A.; Edeling, Melissa A.; Johnson, Syd; Pierson, Theodore C.; Diamond, Michael S.; Fremont, Daved H.

2012-01-01

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development. PMID:23055922

Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

PubMed Central

Purtha, Whitney E.; Tedder, Thomas F.; Johnson, Syd

2011-01-01

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) persist after clearance of infection, yet the specific and nonredundant role MBCs play in subsequent protection is unclear. After resolution of West Nile virus infection in mice, we demonstrate that LLPCs were specific for a single dominant neutralizing epitope, such that immune serum poorly inhibited a variant virus that encoded a mutation at this critical epitope. In contrast, a large fraction of MBC produced antibody that recognized both wild-type (WT) and mutant viral epitopes. Accordingly, antibody produced by the polyclonal pool of MBC neutralized WT and variant viruses equivalently. Remarkably, we also identified MBC clones that recognized the mutant epitope better than the WT protein, despite never having been exposed to the variant virus. The ability of MBCs to respond to variant viruses in vivo was confirmed by experiments in which MBCs were adoptively transferred or depleted before secondary challenge. Our data demonstrate that class-switched MBC can respond to variants of the original pathogen that escape neutralization of antibody produced by LLPC without a requirement for accumulating additional somatic mutations. PMID:22162833

Epitope Mapping by Phage Display.

PubMed

Moreira, Gustavo Marçal Schmidt Garcia; Fühner, Viola; Hust, Michael

2018-01-01

Among the molecules of the immune system, antibodies, particularly monoclonal antibodies (mAbs), have been shown to be interesting for many biological applications. Due to their ability to recognize only a unique part of their target, mAbs are usually very specific. These targets can have many different compositions, but the most common ones are proteins or peptides that are usually from outside the host, although self-proteins can also be targeted in autoimmune diseases, or in some types of cancer. The parts of a mAb that interact with its target compose the paratope, while the recognized parts of the target compose the epitope. Knowing the epitope is valuable for the improvement of a biological product, e.g., a diagnostic assay, a therapeutic mAb, or a vaccine, as well as for the elucidation of immune responses. The current techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Even though there are several techniques available, each has its pros and cons. Thus, the choice for one of them is usually dependent on the preference and availability of the researcher, opening possibility for improvement, or development of alternative techniques. Phage display, for example, is a versatile technology, which allows the presentation of many different oligopeptides that can be tested against different antibodies, fitting the need for an epitope mapping approach. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue

PubMed Central

Korzeniowska-Kowal, Agnieszka; Kochman, Agata; Gamian, Elżbieta; Lis-Nawara, Anna; Lipiński, Tomasz; Seweryn, Ewa; Ziółkowski, Piotr; Gamian, Andrzej

2015-01-01

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized. PMID:26086646

Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease

PubMed Central

Signorino, Giacomo; Arnaboldi, Paul M.; Petzke, Mary M.

2014-01-01

Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease. PMID:24623628

Identification of broadly reactive epitopes targeting major glycoproteins of Herpes simplex virus (HSV) 1 and 2 - An immunoinformatics analysis.

PubMed

Chauhan, Varun; Goyal, Kapil; Singh, Mini P

2018-07-01

Infections due to both HSV-1 and HSV-2 constitute an enormous health burden worldwide. Development of vaccine against herpes infections is a WHO supported public health priority. The viral glycoproteins have always been the major hotspots for vaccine designing. The present study was aimed to identify the conserved T and B cell epitopes in the major glycoproteins of both HSV-1 and HSV-2 via rigorous computational approaches. Identification of promiscuous T cell epitopes is of utmost importance in vaccine designing as such epitopes are capable of binding to several allelic forms of HLA and could generate effective immune response in the host. The criteria designed for identification of T and B cell epitopes was that it should be conserved in both HSV-1 and 2, promiscuous, have high affinity towards HLA alleles, should be located on the surface of glycoproteins and not be present in the glycosylation sites. This study led to the identification of 17 HLA Class II and 26 HLA Class I T cell epitopes, 9 linear and some conformational B cell epitopes. The identified T cell epitopes were further subjected to molecular docking analysis to analyze their binding patterns. Altogether we have identified 4 most promising regions in glycoproteins (2-gB, 1-gD, 1-gH) of HSV-1 and 2 which are promiscuous to HLA Class II alleles and have overlapping HLA Class I and B cell epitopes, which could be very useful in generating both arms of immune response in the host i.e. adaptive as well as humoral immunity. Further the authors propose the cross-validation of the identified epitopes in experimental settings for confirming their immunogenicity to support the present findings. Copyright © 2018 Elsevier B.V. All rights reserved.

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referredmore » to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.« less

Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1.

PubMed

Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

2013-04-30

The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106-120, 166-180, 289-300 and 313-332). We have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents.

Blister-inducing antibodies target multiple epitopes on collagen VII in mice

PubMed Central

Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

2014-01-01

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

Dissecting Antibodies with Regards to Linear and Conformational Epitopes

PubMed Central

Forsström, Björn; Bisławska Axnäs, Barbara; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

2015-01-01

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension.

PubMed

Ihalainen, Teemu O; Aires, Lina; Herzog, Florian A; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

2015-12-01

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension

NASA Astrophysics Data System (ADS)

Ihalainen, Teemu O.; Aires, Lina; Herzog, Florian A.; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

2015-12-01

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

Identification of linear B-cell epitopes on goose parvovirus non-structural protein.

PubMed

Yu, Tian-Fei; Ma, Bo; Wang, Jun-Wei

2016-10-15

Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. Copyright © 2016. Published by Elsevier B.V.

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

PubMed

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C Ronald; Sidhu, Sachdev S

2017-05-19

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

PubMed Central

Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

2013-01-01

CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

A DNA Aptamer Recognizes the Asp f 1 Allergen of Aspergillus fumigatus

PubMed Central

Low, Swee Yang; Hill, Jane E.; Peccia, Jordan

2009-01-01

Allergies are caused by the binding of IgE antibodies onto specific sites on allergens. However, in the assessment of exposure to airborne allergens, current techniques such as whole spore counts fail to account for the presence of these allergenic epitopes that trigger allergic reactions. The objective of the research is to develop a DNA aptamer for the Asp f 1 allergen of the pathogenic fungus Aspergillus fumigatus, using an IgE-binding epitope of the allergen as the target for aptamer selection. Through in vitro SELEX, an aptamer has been produced that binds with nanomolar affinity to the Asp f 1 IgE-epitope. The aptamer is also able to recognize the native Asp f 1 allergen, and does not bind to allergenic proteins from non-target mold species such as Alternaria alternata. Production of this aptamer provides proof-of-principle that allergen measurement methods can be developed to indicate the potent fraction, or allergenicity, of allergens. PMID:19545545

Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120

PubMed Central

Kong, Leopold; Lee, Jeong Hyun; Doores, Katie J.; Murin, Charles D.; Julien, Jean-Philippe; McBride, Ryan; Liu, Yan; Marozsan, Andre; Cupo, Albert; Klasse, Per-Johan; Hoffenberg, Simon; Caulfield, Michael; King, C. Richter; Hua, Yuanzi; Le, Khoa M.; Khayat, Reza; Deller, Marc C.; Clayton, Thomas; Tien, Henry; Feizi, Ten; Sanders, Rogier W.; Paulson, James C.; Moore, John P.; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.

2013-01-01

A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization. PMID:23708606

Mapping of the antigenic determinants of the T. cruzi kinetoplastid membrane protein-11. Identification of a linear epitope specifically recognized by human Chagasic sera.

PubMed

Thomas, M C; Longobardo, M V; Carmelo, E; Marañón, C; Planelles, L; Patarroyo, M E; Alonso, C; López, M C

2001-03-01

The high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino- and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease.

Mapping of the antigenic determinants of the T. cruzi kinetoplastid membrane protein-11. Identification of a linear epitope specifically recognized by human Chagasic sera

PubMed Central

Thomas, M C; Longobardo, M V; Carmelo, E; Marañón, C; Planelles, L; Patarroyo, M E; Alonso, C; López, M C

2001-01-01

The high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino- and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease. PMID:11298135

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

PubMed Central

Earnshaw, W C; Machlin, P S; Bordwell, B J; Rothfield, N F; Cleveland, D W

1987-01-01

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting. Images PMID:2440036

Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B.

PubMed

Earnshaw, W C; Machlin, P S; Bordwell, B J; Rothfield, N F; Cleveland, D W

1987-07-01

A cDNA clone encoding CENP-B, the 80-kDa human centromere autoantigen, was used to construct a panel of hybrid proteins containing four different regions of CENP-B. These have allowed us to identify three independent epitopes on CENP-B that are targets of autoantibodies. Two of these are recognized concurrently in greater than or equal to 90% of patient sera containing anticentromere autoantibodies (ACA), conclusively demonstrating that this autoimmune response is polyclonal. When present and previous data are combined, ACA are shown to recognize at least five independent epitopes on CENP-B. A radioimmunoassay based on cloned CENP-B has demonstrated that sera from greater than or equal to 96% of patients with ACA recognize the cloned antigen, thus defining a region of the protein that is recognized by virtually all patients with ACA. These findings have significant implications for models that seek to explain the origin of ACA and for the future detection of this group of autoantibodies in the clinical setting.

Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein–Barr virus as a potential vaccine and diagnostic agent

PubMed Central

Lin, Xiaoyun; Chen, Shao; Xue, Xiangyang; Lu, Lijun; Zhu, Shanli; Li, Wenshu; Chen, Xiangmin; Zhong, Xiaozhi; Jiang, Pengfei; Sename, Torsoo Sophia; Zheng, Yi; Zhang, Lifang

2016-01-01

Epstein–Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients. PMID:25864917

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

USGS Publications Warehouse

Weins, G.; Chien, M.S.; Winton, J.R.; Kaatari, S.L.

1999-01-01

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA. the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.

Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

PubMed Central

Cannella, Anthony P.; Arlehamn, Cecilia S. Lindestam; Sidney, John; Patra, Kailash P.; Torres, Katherine; Tsolis, Renee M.; Liang, Li; Felgner, Philip L.; Saito, Mayuko; Gotuzzo, Eduardo; Gilman, Robert H.; Sette, Alessandro

2014-01-01

Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen. PMID:24126518

Ana o 2, a major cashew (Anacardium occidentale L.) nut allergen of the legumin family.

PubMed

Wang, Fang; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

2003-09-01

We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen. A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology. Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes. We conclude that this legumin-like protein is a major allergen in cashew nut. Copyright 2003 S. Karger AG, Basel

Utilizing nanobody technology to target non-immunodominant domains of VAR2CSA.

PubMed

Ditlev, Sisse B; Florea, Raluca; Nielsen, Morten A; Theander, Thor G; Magez, Stefan; Boeuf, Philippe; Salanti, Ali

2014-01-01

Placental malaria is a major health problem for both pregnant women and their fetuses in malaria endemic regions. It is triggered by the accumulation of Plasmodium falciparum-infected erythrocytes (IE) in the intervillous spaces of the placenta and is associated with foetal growth restriction and maternal anemia. IE accumulation is supported by the binding of the parasite-expressed protein VAR2CSA to placental chondroitin sulfate A (CSA). Defining specific CSA-binding epitopes of VAR2CSA, against which to target the immune response, is essential for the development of a vaccine aimed at blocking IE adhesion. However, the development of a VAR2CSA adhesion-blocking vaccine remains challenging due to (i) the large size of VAR2CSA and (ii) the extensive immune selection for polymorphisms and thereby non-neutralizing B-cell epitopes. Camelid heavy-chain-only antibodies (HcAbs) are known to target epitopes that are less immunogenic to classical IgG and, due to their small size and protruding antigen-binding loop, able to reach and recognize cryptic, conformational epitopes which are inaccessible to conventional antibodies. The variable heavy chain (VHH) domain is the antigen-binding site of camelid HcAbs, the so called Nanobody, which represents the smallest known (15 kDa) intact, native antigen-binding fragment. In this study, we have used the Nanobody technology, an approach new to malaria research, to generate small and functional antibody fragments recognizing unique epitopes broadly distributed on VAR2CSA.

The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5.

PubMed

Huarte, Nerea; Lorizate, Maier; Maeso, Rubén; Kunert, Renate; Arranz, Rocio; Valpuesta, José M; Nieva, José L

2008-09-01

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing, Clustering, and Characterizing a Tool Box*

PubMed Central

Zhao, Min; Li, Xiao-Jing; Tang, Zi-Min; Yang, Fan; Wang, Si-Ling; Cai, Wei; Zhang, Ke; Xia, Ning-Shao; Zheng, Zi-Zheng

2015-01-01

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process. PMID:26085097

Identification of conserved and HLA-A*2402-restricted epitopes in Dengue virus serotype 2.

PubMed

Duan, Zhi-Liang; Liu, Hui-Fang; Huang, Xi; Wang, Si-Na; Yang, Jin-Lin; Chen, Xin-Yu; Li, De-Zhou; Zhong, Xiao-Zhi; Chen, Bo-Kun; Wen, Jin-Sheng

2015-01-22

In this study, we set out to identify dengue virus serotype 2 (DENV-2)-specific HLA-A*2402-restricted epitopes and determine the characteristics of T cells generated to these epitopes. We screened the full-length amino-acid sequence of DENV-2 to find potential epitopes using the SYFPEITHI algorithm. Twelve putative HLA-A*2402-binding peptides conserved in hundreds of DENV-2 strains were synthesized, and the HLA restriction of peptides was tested in HLA-A*2402 transgenic mice. Nine peptides (NS4b(228-237), NS2a(73-81), E(298-306), M(141-149), NS4a(96-105), NS4b(159-168), NS5(475-484), NS1(162-171), and NS5(611-620)) induced high levels of peptide-specific IFN-γ-secreting cells in HLA-A*2402 transgenic mice. Apart from IFN-γ, NS4b(228-237-), NS2a(73-81-) and E(298-306)-specific CD8(+) cells produced TNF-α and IL-6 simultaneously, whereas M(141-149-) and NS5(475-484-) CD8(+) cells produced only IL-6. Moreover, splenic mononuclear cells (SMCs) efficiently recognized and killed peptide-pulsed splenocytes. Furthermore, each of nine peptides could be recognized by splenocytes from DENV-2-infected HLA-A*2402 transgenic mice. The SMCs from HLA-A*2402 transgenic mice immunized with nine immunogenic peptides efficiently killed DENV-2-infected splenic monocytes. The present identified epitopes have the potential to be new diagnostic tools for characterization of T-cell immunity in DENV infection and may serve as part of a universal epitope-based vaccine. Copyright © 2014 Elsevier B.V. All rights reserved.

Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes.

PubMed

McMurtrey, Curtis P; Lelic, Alina; Piazza, Paolo; Chakrabarti, Ayan K; Yablonsky, Eric J; Wahl, Angela; Bardet, Wilfried; Eckerd, Annette; Cook, Robert L; Hess, Rachael; Buchli, Rico; Loeb, Mark; Rinaldo, Charles R; Bramson, Jonathan; Hildebrand, William H

2008-02-26

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.

Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

PubMed

Yamada, Shinji; Kaneko, Mika K; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Ogasawara, Satoshi; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Harada, Hiroyuki; Kato, Yukinari

2018-04-02

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

A HER2-specific Modified Fc Fragment (Fcab) Induces Antitumor Effects Through Degradation of HER2 and Apoptosis

PubMed Central

Leung, Kin-Mei; Batey, Sarah; Rowlands, Robert; Isaac, Samine J; Jones, Phil; Drewett, Victoria; Carvalho, Joana; Gaspar, Miguel; Weller, Sarah; Medcalf, Melanie; Wydro, Mateusz M; Pegram, Robert; Mudde, Geert C; Bauer, Anton; Moulder, Kevin; Woisetschläger, Max; Tuna, Mihriban; Haurum, John S; Sun, Haijun

2015-01-01

FS102 is a HER2-specific Fcab (Fc fragment with antigen binding), which binds HER2 with high affinity and recognizes an epitope that does not overlap with those of trastuzumab or pertuzumab. In tumor cells that express high levels of HER2, FS102 caused profound HER2 internalization and degradation leading to tumor cell apoptosis. The antitumor effect of FS102 in patient-derived xenografts (PDXs) correlated strongly with the HER2 amplification status of the tumors. Superior activity of FS102 over trastuzumab or the combination of trastuzumab and pertuzumab was observed in vitro and in vivo when the gene copy number of HER2 was equal to or exceeded 10 per cell based on quantitative polymerase chain reaction (qPCR). Thus, FS102 induced complete and sustained tumor regression in a significant proportion of HER2-high PDX tumor models. We hypothesize that the unique structure and/or epitope of FS102 enables the Fcab to internalize and degrade cell surface HER2 more efficiently than standard of care antibodies. In turn, increased depletion of HER2 commits the cells to apoptosis as a result of oncogene shock. FS102 has the potential of a biomarker-driven therapeutic that derives superior antitumor effects from a unique mechanism-of-action in tumor cells which are oncogenically addicted to the HER2 pathway due to overexpression. PMID:26234505

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

PubMed

Eikawa, Shingo; Kakimi, Kazuhiro; Isobe, Midori; Kuzushima, Kiyotaka; Luescher, Immanuel; Ohue, Yoshihiro; Ikeuchi, Kazuhiro; Uenaka, Akiko; Nishikawa, Hiroyoshi; Udono, Heiichiro; Oka, Mikio; Nakayama, Eiichi

2013-01-15

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response. Copyright © 2012 UICC.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

PubMed Central

Hilton, Hugo G; Parham, Peter

2013-01-01

Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing 3-6 HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with one of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1μg/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50μg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. PMID:23510417

Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

PubMed

Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

2014-01-01

B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use.

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

PubMed

Hong, Kwang-Won; Kim, Chang-Goo; Lee, Seung-Hyun; Chang, Ki-Hwan; Shin, Yong Won; Ryoo, Kyung-Hwan; Kim, Se-Ho; Kim, Yong-Sung

2010-01-01

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms.

PubMed Central

MacInnes, J I; Rosendal, S

1987-01-01

Outer membrane protein (OMP)-enriched extracts and whole-cell protein preparations of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms were examined by polyacrylamide gel electrophoresis and immunoblotting. Both the OMP-enriched and whole-cell protein profiles of Actinobacillus suis, A. pleuropneumoniae (NAD-independent biovar), A. lignieresii, and Pasteurella haemolytica were very similar to those of H. pleuropneumoniae serotypes 1 to 8. Antisera prepared against H. pleuropneumoniae typically recognized three major OMP antigens with approximate molecular weights of 17,000 (17K), 32K, and 42K in immunoblots of H. pleuropneumoniae serotypes 1 to 8, Actinobacillus spp., and P. haemolytica. Antisera prepared against Actinobacillus spp. and Haemophilus sp. "minor group" also recognized these 17K, 32K, and 42K antigens. Using absorbed sera, we demonstrated that the 17K antigen had an epitope (or epitopes) common to all the gram-negative organisms examined, including Escherichia coli. The 32K and 42K antigens had epitopes common to members of the family Pasteurellaceae but, in the case of the 32K antigen, also contained unique epitopes. These results provide a basis for understanding the lack of specificity of serodiagnostic tests for H. pleuropneumoniae infection and provide another line of evidence for the association of H. pleuropneumoniae with the genus Actinobacillus. Images PMID:3298061

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots.

PubMed Central

Freshour, G.; Clay, R. P.; Fuller, M. S.; Albersheim, P.; Darvill, A. G.; Hahn, M. G.

1996-01-01

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner. PMID:12226270

Hepatitis C virus Broadly Neutralizing Monoclonal Antibodies Isolated 25 Years after Spontaneous Clearance.

PubMed

Merat, Sabrina J; Molenkamp, Richard; Wagner, Koen; Koekkoek, Sylvie M; van de Berg, Dorien; Yasuda, Etsuko; Böhne, Martino; Claassen, Yvonne B; Grady, Bart P; Prins, Maria; Bakker, Arjen Q; de Jong, Menno D; Spits, Hergen; Schinkel, Janke; Beaumont, Tim

2016-01-01

Hepatitis C virus (HCV) is world-wide a major cause of liver related morbidity and mortality. No vaccine is available to prevent HCV infection. To design an effective vaccine, understanding immunity against HCV is necessary. The memory B cell repertoire was characterized from an intravenous drug user who spontaneously cleared HCV infection 25 years ago. CD27+IgG+ memory B cells were immortalized using BCL6 and Bcl-xL. These immortalized B cells were used to study antibody-mediated immunity against the HCV E1E2 glycoproteins. Five E1E2 broadly reactive antibodies were isolated: 3 antibodies showed potent neutralization of genotype 1 to 4 using HCV pseudotyped particles, whereas the other 2 antibodies neutralized genotype 1, 2 and 3 or 1 and 2 only. All antibodies recognized non-linear epitopes on E2. Finally, except for antibody AT12-011, which recognized an epitope consisting of antigenic domain C /AR2 and AR5, all other four antibodies recognized epitope II and domain B. These data show that a subject, who spontaneously cleared HCV infection 25 years ago, still has circulating memory B cells that are able to secrete broadly neutralizing antibodies. Presence of such memory B cells strengthens the argument for undertaking the development of an HCV vaccine.

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

PubMed

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

PubMed

Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

1997-06-01

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1

PubMed Central

2013-01-01

Background The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Findings Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106–120, 166–180, 289–300 and 313–332). Conclusions We have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents. PMID:23631709

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Hua; Jiang Lifang; Fang Danyun

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosenmore » for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.« less

Sera of children with hepatitis C infection and anti-liver-kidney microsome-1 antibodies recognize different CYP2D6 epitopes than adults with LKM+/HCV+ sera.

PubMed

Herzog, D; Yamamoto, A M; Jara, P; Maggiore, G; Sarles, J; Alvarez, F

1999-11-01

Liver-kidney microsome type 1 (LKM1) antibodies are specific markers of autoimmune hepatitis (AIH) type 2. Antibodies to LKM1 have been found in 2% to 3% of adults infected with hepatitis C virus (HCV) without AIH. Thirty percent of these antibodies are directed against linear sequences of CYP2D6 protein. LKM1 antibodies in HCV+/LKM1+ sera and in sera of AIH patients do not recognize the same CYP2D6 epitopes. The current study was conducted to determine whether LKM1 antibodies in HCV+/LKM1+ children's sera are the result of the same immune response as the antibodies described in AIH type 2 and in HCV+/LKM1+ adult patients. Sera from 10 HCV+/LKM1+ children were tested against human liver microsomal and cytosolic proteins by Western blot analysis and against synthetic peptides of the CYP2D6 sequence between amino acids 200 and 429 by dot blot. The same sera were tested against radiolabeled CYP2D6 by immunoprecipitation. Four of 10 sera tested by Western blot analysis showed immunoglobulin (Ig) G-type antibodies against CYP2D6, and 2 had antibodies against proteins of 58, 66, and 84 kDa. One of the sera also contained IgM-type anti-66-kDa and 84-kDa proteins. The radioligand test detected anti-CYP2D6 antibodies in 9 of 10 patients. Five of the anti-CYP2D6-positive sera recognized a peptide between amino acids 200 and 429 including amino acids 254-271. Most HCV+/LKM1+ sera from children recognize conformational epitopes of the CYP2D6 antigen, and half recognize linear epitopes. Some HCV+/LKM1+ sera demonstrated antibodies against the AIH type 2 main antigenic site of the CYP2D6. Screening of HCV RNA should be performed before starting treatment of presumed autoimmune hepatitis associated with LKM1.

Therapeutic Vaccination against the Rhesus Lymphocryptovirus EBNA-1 Homologue, rhEBNA-1, Elicits T Cell Responses to Novel Epitopes in Rhesus Macaques

PubMed Central

Silveira, Eduardo L. V.; Fogg, Mark H.; Leskowitz, Rachel M.; Ertl, Hildegund C.; Wiseman, Roger W.; O'Connor, David H.; Lieberman, Paul; Wang, Fred

2013-01-01

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC–rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination. PMID:24089556

Influence of an immunodominant herpes simplex virus type 1 CD8+ T cell epitope on the target hierarchy and function of subdominant CD8+ T cells

PubMed Central

2017-01-01

Herpes simplex virus type 1 (HSV-1) latency in sensory ganglia such as trigeminal ganglia (TG) is associated with a persistent immune infiltrate that includes effector memory CD8+ T cells that can influence HSV-1 reactivation. In C57BL/6 mice, HSV-1 induces a highly skewed CD8+ T cell repertoire, in which half of CD8+ T cells (gB-CD8s) recognize a single epitope on glycoprotein B (gB498-505), while the remainder (non-gB-CD8s) recognize, in varying proportions, 19 subdominant epitopes on 12 viral proteins. The gB-CD8s remain functional in TG throughout latency, while non-gB-CD8s exhibit varying degrees of functional compromise. To understand how dominance hierarchies relate to CD8+ T cell function during latency, we characterized the TG-associated CD8+ T cells following corneal infection with a recombinant HSV-1 lacking the immunodominant gB498-505 epitope (S1L). S1L induced a numerically equivalent CD8+ T cell infiltrate in the TG that was HSV-specific, but lacked specificity for gB498-505. Instead, there was a general increase of non-gB-CD8s with specific subdominant epitopes arising to codominance. In a latent S1L infection, non-gB-CD8s in the TG showed a hierarchy targeting different epitopes at latency compared to at acute times, and these cells retained an increased functionality at latency. In a latent S1L infection, these non-gB-CD8s also display an equivalent ability to block HSV reactivation in ex vivo ganglionic cultures compared to TG infected with wild type HSV-1. These data indicate that loss of the immunodominant gB498-505 epitope alters the dominance hierarchy and reduces functional compromise of CD8+ T cells specific for subdominant HSV-1 epitopes during viral latency. PMID:29206240

Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

PubMed

Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

2012-07-01

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

PubMed Central

Xing, Dongxia; Li, Sufang; Robinson, Simon N.; Yang, Hong; Steiner, David; Komanduri, Krishna V.; Shpall, Elizabeth J.

2009-01-01

In the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these “Th-1 DCs” exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8+ T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies. PMID:19171878

Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

PubMed Central

Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

2016-01-01

Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

PubMed

Hertz, Tomer; Ahmed, Hasan; Friedrich, David P; Casimiro, Danilo R; Self, Steven G; Corey, Lawrence; McElrath, M Juliana; Buchbinder, Susan; Horton, Helen; Frahm, Nicole; Robertson, Michael N; Graham, Barney S; Gilbert, Peter

2013-01-01

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

PubMed

Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

2005-06-01

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

PubMed

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

2017-11-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Marie; Mine, Yoshinori

The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e.,more » A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-{gamma} and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.« less

Greater epitope recognition of shrimp allergens by children than by adults suggests that shrimp sensitization decreases with age.

PubMed

Ayuso, Rosalía; Sánchez-Garcia, Silvia; Lin, Jing; Fu, Zhiyan; Ibáñez, María Dolores; Carrillo, Teresa; Blanco, Carlos; Goldis, Marina; Bardina, Ludmila; Sastre, Joaquín; Sampson, Hugh A

2010-06-01

Shellfish allergy is a long-lasting disorder typically affecting adults. Despite its high prevalence, there is limited information about allergenic shrimp proteins and the epitopes implicated in such allergic reactions. We sought to identify the IgE-binding epitopes of the 4 shrimp allergens and to characterize epitope recognition profiles of children and adults with shrimp allergy. Fifty-three subjects, 34 children and 19 adults, were selected with immediate allergic reactions to shrimp, increased shrimp-specific serum IgE levels, and positive immunoblot binding to shrimp. Study subjects and 7 nonatopic control subjects were tested by means of peptide microarray for IgE binding with synthetic overlapping peptides spanning the sequences of Litopenaeus vannamei shrimp tropomyosin, arginine kinase (AK), myosin light chain (MLC), and sarcoplasmic calcium-binding protein (SCP). The Wilcoxon test was used to determine significant differences in z scores between patients and control subjects. The median shrimp IgE level was 4-fold higher in children than in adults (47 vs 12.5 kU(A)/L). The frequency of allergen recognition was higher in children (tropomyosin, 81% [94% for children and 61% for adults]; MLC, 57% [70% for children and 31% for adults]; AK, 51% [67% for children and 21% for adults]; and SCP, 45% [59% for children and 21% for adults]), whereas control subjects showed negligible binding. Seven IgE-binding regions were identified in tropomyosin by means of peptide microarray, confirming previously identified shrimp epitopes. In addition, 3 new epitopes were identified in tropomyosin (epitopes 1, 3, and 5b-c), 5 epitopes were identified in MLC, 3 epitopes were identified in SCP, and 7 epitopes were identified in AK. Interestingly, frequency of individual epitope recognition, as well as intensity of IgE binding, was significantly greater in children than in adults for all 4 proteins. Children with shrimp allergy have greater shrimp-specific IgE antibody levels and show more intense binding to shrimp peptides and greater epitope diversity than adults. Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

PubMed Central

Alonso-Urmeneta, B.; Marín, C.; Aragón, V.; Blasco, J. M.; Díaz, R.; Moriyón, I.

1998-01-01

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen. PMID:9801329

Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

PubMed Central

2012-01-01

Background To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents. PMID:22264266

HIV Molecular Immunology 2014

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yusim, Karina; Korber, Bette Tina Marie; Barouch, Dan

HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2014 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and binding sites within each protein, moving from left to right through themore » coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as crossreactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins are provided.« less

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

PubMed Central

Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

2015-01-01

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

PubMed

Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

2015-09-09

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

In silico analyses of structural and allergenicity features of sapodilla (Manilkara zapota) acidic thaumatin-like protein in comparison with allergenic plant TLPs.

PubMed

Ashok Kumar, Hassan G; Venkatesh, Yeldur P

2014-02-01

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related family (PR-5) of plant defense proteins. TLPs from only 32 plant genera have been identified as pollen or food allergens. IgE epitopes on allergens play a central role in food allergy by initiating cross-linking of specific IgE on basophils/mast cells. A comparative analysis of pollen- and food-allergenic TLPs is lacking. The main objective of this investigation was to study the structural and allergenicity features of sapodilla (Manilkara zapota) acidic TLP (TLP 1) by in silico methods. The allergenicity prediction of composite sequence of sapodilla TLP 1 (NCBI B3EWX8.1, G5DC91.1) was performed using FARRP, Allermatch and Evaller web tools. A homology model of the protein was generated using banana TLP template (1Z3Q) by HHPRED-MODELLER. B-cell linear epitope prediction was performed using BCpreds and BepiPred. Sapodilla TLP 1 matched significantly with allergenic TLPs from olive, kiwi, bell pepper and banana. IgE epitope prediction as performed using AlgPred indicated the presence of 2 epitopes (epitope 1: residues 36-48; epitope 2: residues 51-63), and a comprehensive analysis of all allergenic TLPs displayed up to 3 additional epitopes on other TLPs. It can be inferred from these analyses that plant allergenic TLPs generally carry 2-3 IgE epitopes. ClustalX alignments of allergenic TLPs indicate that IgE epitopes 1 and 2 are common in food allergenic TLPs, and IgE epitopes 2 and 3 are common in pollen allergenic TLPs; IgE epitope 2 overlaps with a portion of the thaumatin family signature. The secondary structural elements of TLPs vary markedly in regions 1 and 2 which harbor all the predicted IgE epitopes in all food and pollen TLPs in either of the region. Further, based on the number of IgE epitopes, food TLPs are grouped into rosid and non-rosid clades. The number and distribution of the predicted IgE epitopes among the allergenic TLPs may explain the specificity of food or pollen allergy as well as the varied degree of cross-reactivity among plant foods and/or pollens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1.

PubMed

Gohain, Neelakshi; Tolbert, William D; Acharya, Priyamvada; Yu, Lei; Liu, Tongyun; Zhao, Pingsen; Orlandi, Chiara; Visciano, Maria L; Kamin-Lewis, Roberta; Sajadi, Mohammad M; Martin, Loïc; Robinson, James E; Kwong, Peter D; DeVico, Anthony L; Ray, Krishanu; Lewis, George K; Pazgier, Marzena

2015-09-01

Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered β-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting--a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses. HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected nonhuman primates (NHPs), and HIV-1-infected humanized mouse models, passive transfer studies in infants born to HIV-infected mothers, and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with postinfection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the nonneutralizing CD4-dependent epitopes in the gp41-reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

PubMed Central

Rosendahl Huber, S. K.; Camps, M. G. M.; Jacobi, R. H. J.; Mouthaan, J.; van Dijken, H.; van Beek, J.; Ossendorp, F.; de Jonge, J.

2015-01-01

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks. PMID:26046664

Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression

PubMed Central

Lu, Yong-Chen; Yao, Xin; Li, Yong F.; El-Gamil, Mona; Dudley, Mark E.; Yang, James C.; Almeida, Jorge R.; Douek, Daniel C.; Samuels, Yardena; Rosenberg, Steven A.; Robbins, Paul F.

2013-01-01

Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the antigen targets recognized by effective melanoma reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses ongoing beyond seven years following adoptive TILs transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific antigen can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated antigens. PMID:23690473

A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

PubMed

Wang, Xiaoyu; Zhou, Defang; Wang, Guihua; Huang, Libo; Zheng, Qiankun; Li, Chengui; Cheng, Ziqiang

2017-12-04

The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

PubMed

Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

2016-02-01

Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner. Copyright © 2015 Elsevier Ltd. All rights reserved.

Recognition pattern of thyroglobulin autoantibody from hypothyroid dogs to tryptic peptides of canine thyroglobulin.

PubMed

Tani, Hiroyuki; Shimizu, Reiko; Sasai, Kazumi; Baba, Eiichiroh

2003-10-01

Circulating thyroglobulin autoantibody (TgAA) was analyzed using the Western immunoblot for determination of the dominant epitopes recognized by TgAA on tryptic peptides of canine thyroglobulin (cTg) in hypothyroid dogs. TgAA was measured in hypothyroid dogs, non-hypothyroid dogs with skin diseases and clinically normal dogs. Five of the 7 hypothyroid dogs, 1 of the 8 dogs with skin diseases and 1 of the 4 normal dogs were positive for TgAA. Four of the 5 TgAA-positive hypothyroid dogs were Golden Retrievers, and 3 of them showed high antibody titers. The sera of TgAA positive-dogs reacted to several peptides, and their patterns varied from sample to sample. Sera from 3 dogs with high titers of TgAA reacted broadly to high molecular weight peptides ranging from 45 to 90 kDa. These Western immunoblot patterns of the sera were disappeared after pretreatment with sufficient amount of intact cTg. All serum samples of both TgAA positive dogs and negative controls reacted to low molecular weight peptides ranging from 15 to 20 kDa. These immunoblot patterns of the sera were not disappeared even after pretreatment with sufficient amount of intact cTg. These findings show the possibility that the epitopes recognized by TgAA depend upon individual dogs with hypothyroidism and these autoantibodies recognize conformational epitopes on the cTg molecule.

Identification of epitopes on nonstructural protein 7 of porcine reproductive and respiratory syndrome virus recognized by monoclonal antibodies using phage-display technology.

PubMed

Wang, Heng; Liu, Rongchang; Zhang, Weidong; Sun, Lingshuang; Ning, Zhangyong; Ji, Fangxiao; Cui, Jin; Zhang, Guihong

2017-08-01

Nonstructural protein 7 (nsp7) of porcine reproductive and respiratory syndrome virus (PRRSV) is considered to be a suitable reagent for the development of serological diagnostic assays. It can be expressed as a soluble recombinant protein in Escherichia coli, and its antibody response may continue up to 202 days post-infection. Furthermore, the region encoded by nsp7 is highly homologous among various strains within the genotype, and the results of nsp7-based enzyme-linked immunosorbent assay (ELISA) showed high agreement with previous Idexx ELISA results. All these evidences suggest the existence of important epitopes on nsp7, though the characteristics of these epitopes remain unclear. In the present study, we prepared three monoclonal antibodies against nsp7 protein and used them to screen the epitope-distribution characteristics of PRRSV nsp7 protein by phage-display technology. We identified a linear epitope NAWGDEDRLN at amino acids 153-162 type II PRRSV nsp7β subunit. This newly defined epitope showed excellent reactivity with PRSSV-positive serum samples. These results further our understanding of the antigenic structure of nsp7 protein, and provide efficient reagents for PRRSV serological tests.

Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans

PubMed Central

Chou, Meng-Yun; Fogelstrand, Linda; Hartvigsen, Karsten; Hansen, Lotte F.; Woelkers, Douglas; Shaw, Peter X.; Choi, Jeomil; Perkmann, Thomas; Bäckhed, Fredrik; Miller, Yury I.; Hörkkö, Sohvi; Corr, Maripat; Witztum, Joseph L.; Binder, Christoph J.

2009-01-01

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis. PMID:19363291

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

PubMed

Seppo, A E; Savilahti, E M; Berin, M C; Sampson, H A; Järvinen, K M

2017-10-01

We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). We measured specific IgA to a series of overlapping peptides in major CM allergens (α s1 -, α s2 -, β- and κ-caseins and β-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in α s1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated. © 2017 John Wiley & Sons Ltd.

Potential contribution of a novel Tax epitope-specific CD4+ T cells to graft-versus-Tax effect in adult T cell leukemia patients after allogeneic hematopoietic stem cell transplantation.

PubMed

Tamai, Yotaro; Hasegawa, Atsuhiko; Takamori, Ayako; Sasada, Amane; Tanosaki, Ryuji; Choi, Ilseung; Utsunomiya, Atae; Maeda, Yasuhiro; Yamano, Yoshihisa; Eto, Tetsuya; Koh, Ki-Ryang; Nakamae, Hirohisa; Suehiro, Youko; Kato, Koji; Takemoto, Shigeki; Okamura, Jun; Uike, Naokuni; Kannagi, Mari

2013-04-15

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for adult T cell leukemia/lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). We previously reported that Tax-specific CD8(+) cytotoxic T lymphocyte (CTL) contributed to graft-versus-ATL effects in ATL patients after allo-HSCT. However, the role of HTLV-1-specific CD4(+) T cells in the effects remains unclear. In this study, we showed that Tax-specific CD4(+) as well as CD8(+) T cell responses were induced in some ATL patients following allo-HSCT. To further analyze HTLV-1-specific CD4(+) T cell responses, we identified a novel HLA-DRB1*0101-restricted epitope, Tax155-167, recognized by HTLV-1-specific CD4(+) Th1-like cells, a major population of HTLV-1-specific CD4(+) T cell line, which was established from an ATL patient at 180 d after allo-HSCT from an unrelated seronegative donor by in vitro stimulation with HTLV-1-infected cells from the same patient. Costimulation of PBMCs with both the identified epitope (Tax155-167) and known CTL epitope peptides markedly enhanced the expansion of Tax-specific CD8(+) T cells in PBMCs compared with stimulation with CTL epitope peptide alone in all three HLA-DRB1*0101(+) patients post-allo-HSCT tested. In addition, direct detection using newly generated HLA-DRB1*0101/Tax155-167 tetramers revealed that Tax155-167-specific CD4(+) T cells were present in all HTLV-1-infected individuals tested, regardless of HSCT. These results suggest that Tax155-167 may be the dominant epitope recognized by HTLV-1-specific CD4(+) T cells in HLA-DRB1*0101(+)-infected individuals and that Tax-specific CD4(+) T cells may augment the graft-versus-Tax effects via efficient induction of Tax-specific CD8(+) T cell responses.

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

PubMed

Hu, Hongbo; Li, Li; Kao, Richard Y; Kou, Binbin; Wang, Zhanguo; Zhang, Liang; Zhang, Huiyuan; Hao, Zhiyong; Tsui, Wayne H; Ni, Anping; Cui, Lianxian; Fan, Baoxing; Guo, Feng; Rao, Shuan; Jiang, Chengyu; Li, Qian; Sun, Manji; He, Wei; Liu, Gang

2005-01-01

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes

PubMed Central

Ruwona, Tinashe B; Mcbride, Ryan; Chappel, Rebecca; Head, Steven R; Ordoukhanian, Phillip; Burton, Dennis R.; Law, Mansun

2014-01-01

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research. PMID:24269751

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva▿

PubMed Central

Graham, Simon P.; Pellé, Roger; Yamage, Mat; Mwangi, Duncan M.; Honda, Yoshikazu; Mwakubambanya, Ramadhan S.; de Villiers, Etienne P.; Abuya, Evelyne; Awino, Elias; Gachanja, James; Mbwika, Ferdinand; Muthiani, Anthony M.; Muriuki, Cecelia; Nyanjui, John K.; Onono, Fredrick O.; Osaso, Julius; Riitho, Victor; Saya, Rosemary M.; Ellis, Shirley A.; McKeever, Declan J.; MacHugh, Niall D.; Gilbert, Sarah C.; Audonnet, Jean-Christophe; Morrison, W. Ivan; van der Bruggen, Pierre; Taracha, Evans L. N.

2008-01-01

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes. PMID:18070892

The analysis with monoclonal antibodies of the heterogeneity of Ia glycoproteins on chronic lymphocytic leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Addis, J.B.; Tisch, R.; Falk, J.A.

The accessible Ia molecules on the surface of chronic lymphocytic leukemia (CLL) cells were quantitated in the cellular radioimmunoassay with saturating concentrations of monoclonal antibodies. Monoclonal antibody 21w4, like DA/2 antibody, recognizes monomorphic determinants of human Ia antigens.The amount of 21w4 or DA/2 bound to CLL cells derived from eight patients (varying from 2.6 to 13.9 x 10/sup 5/ molecules/cell) appears to be the maximum observed with the antibodies studied. Two other antibodies, 18d5 and 21r5, although also directed at nonpolymorphic Ia determinants, bind differentially to CLL cells, with the ratios of 21r5/21w4 and 18d5/21w4 varying from 0.08 to 0.90.more » Sequential immunoprecipitation studies have established that the four epitopes 18d5, 21r5, 21w4, and DA/2 were present on the same molecules. All Ia molecules express 21w4 and DA/2 epitopes, whereas only certain subsets of Ia molecules carry accessible 21r5 or 18d5 epitopes. Competitive binding studies showed that the epitopes recognized by the four monoclonal antibodies were different. Monoclonal antibodies 21r5 and 21w4 did not inhibit each other's binding. Furthermore, binding of 21w4 to CLL cells potentiated the binding of /sup 125/I-21r5 IgG to the same cells, suggesting that binding of 21w4 antibody induces a conformational change in the molecule that renders 21r5 epitopes more accessible.« less

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

PubMed Central

Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R.; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. PMID:27494183

Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

PubMed

Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

2008-12-01

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

PubMed

Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts

PubMed Central

Maleki, Soheila J.; Teuber, Suzanne S.; Cheng, Hsiaopo; Chen, Deliang; Comstock, Sarah S.; Ruan, Sanbao; Schein, Catherine H.

2011-01-01

Background Cross reactivity between peanuts and tree nuts implies that similar IgE epitopes are present in their proteins. Objective To determine whether walnut sequences similar to known peanut IgE binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins (SDAP), react with IgE from sera of patients with allergy to walnut and/or peanut. Methods Patient sera were characterized by Western blotting for IgE-binding to nut protein extracts, and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive ELISA was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. Results Sequences from the vicilin walnut allergen Jug r 2 which had low PD values to epitopes of the peanut allergen Ara h 2, a 2s-albumin, bound IgE in sera from five patients who reacted to either walnut, peanut or both. A walnut epitope recognized by 6 patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. A predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. Conclusions Sequences with low PD value (<8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens. PMID:21883278

Epitope mapping of PR81 anti-MUC1 monoclonal antibody following PEPSCAN and phage display techniques.

PubMed

Mohammadi, Mohammad; Rasaee, Mohammad Javad; Rajabibazl, Masoumeh; Paknejad, Malihe; Zare, Mehrak; Mohammadzadeh, Sara

2007-08-01

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.

Cryptic Nature of a Conserved, CD4-Inducible V3 Loop Neutralization Epitope in the Native Envelope Glycoprotein Oligomer of CCR5-Restricted, but Not CXCR4-Using, Primary Human Immunodeficiency Virus Type 1 Strains

PubMed Central

Lusso, Paolo; Earl, Patricia L.; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A.; Burastero, Samuele E.

2005-01-01

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies. PMID:15890935

Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein

PubMed Central

Kaever, Thomas; Meng, Xiangzhi; Matho, Michael H.; Schlossman, Andrew; Li, Sheng; Sela-Culang, Inbal; Ofran, Yanay; Buller, Mark; Crump, Ryan W.; Parker, Scott; Frazier, April; Crotty, Shane; Zajonc, Dirk M.; Peters, Bjoern

2014-01-01

ABSTRACT Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies. IMPORTANCE Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice. PMID:25031354

Antibody protection reveals extended epitopes on the human TSH receptor.

PubMed

Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A; Davies, Terry F

2012-01-01

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

PubMed Central

Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M. Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A.; Davies, Terry F.

2012-01-01

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1–412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. PMID:22957097

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

PubMed

McBride, Ryan; Head, Steven R; Ordoukhanian, Phillip; Law, Mansun

2016-01-01

With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.

Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells.

PubMed

Wang, Yufei; Whittall, Trevor; McGowan, Edward; Younson, Justine; Kelly, Charles; Bergmeier, Lesley A; Singh, Mahavir; Lehner, Thomas

2005-03-15

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.

Navigating through the Jungle of Allergens: Features and Applications of Allergen Databases.

PubMed

Radauer, Christian

2017-01-01

The increasing number of available data on allergenic proteins demanded the establishment of structured, freely accessible allergen databases. In this review article, features and applications of 6 of the most widely used allergen databases are discussed. The WHO/IUIS Allergen Nomenclature Database is the official resource of allergen designations. Allergome is the most comprehensive collection of data on allergens and allergen sources. AllergenOnline is aimed at providing a peer-reviewed database of allergen sequences for prediction of allergenicity of proteins, such as those planned to be inserted into genetically modified crops. The Structural Database of Allergenic Proteins (SDAP) provides a database of allergen sequences, structures, and epitopes linked to bioinformatics tools for sequence analysis and comparison. The Immune Epitope Database (IEDB) is the largest repository of T-cell, B-cell, and major histocompatibility complex protein epitopes including epitopes of allergens. AllFam classifies allergens into families of evolutionarily related proteins using definitions from the Pfam protein family database. These databases contain mostly overlapping data, but also show differences in terms of their targeted users, the criteria for including allergens, data shown for each allergen, and the availability of bioinformatics tools. © 2017 S. Karger AG, Basel.

Antibodies to calmodulin during experimental Trypanosoma brucei rhodesiense infections in rabbits.

PubMed Central

Ruben, L; Patton, C L

1985-01-01

Calmodulin is an intracellular Ca2+ receptor protein which regulates a wide variety of enzymatic processes in eukaryotic cells examined in detail. Native calmodulin is not antigenic in rabbits because of its small size, high degree of amino acid sequence conservation and hydrophobicity. African trypanosomes contain a novel calmodulin which is structurally distinct from bovine brain and Tetrahymena calmodulins. In the present study, we examine the antibody response towards these calmodulins during chronic Trypanosoma brucei rhodesiense infections. Injection of purified trypanosome calmodulin into rabbits stimulates the production of specific IgG antibodies which recognize trypanosome, but not bovine brain or Tetrahymena calmodulins. By contrast, during chronic T. brucei infections in rabbits, antibodies (IgG + IgM + IgA) that recognize trypanosome, Tetrahymena and mammalian calmodulins arise. When only IgG antibodies are evaluated from infection sera, the major response is against mammalian and Tetrahymena calmodulins. Significantly fewer IgG antibodies are measured in the infection sera which recognize trypanosome calmodulin, while the non-specific control protein, chicken ovalbumin, is not recognized. Peak IgG antibody responses against calmodulin occur between Days 30-34 post-infection. Competition assays indicate that Tetrahymena and mammalian calmodulins are recognized at identical epitopes which are distinct from epitopes on trypanosome calmodulin. We conclude that, in the context of chronic T. brucei infections in rabbits, antibodies arise which are able to recognize mammalian host calmodulin. Images Figure 1 PMID:2414212

Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

PubMed

Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

2017-11-22

Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

CD147 contains different bioactive epitopes involving the regulation of cell adhesion and lymphocyte activation.

PubMed

Chiampanichayakul, Sawitree; Peng-in, Pakorn; Khunkaewla, Panida; Stockinger, Hannes; Kasinrerk, Watchara

2006-01-01

CD147 is a leukocyte surface molecule which belongs to the immunoglobulin superfamily. It is broadly expressed on various cell types and is a lymphocyte activation-associated molecule. In order to study the function of CD147, five CD147 monoclonal antibodies (mAbs) were generated: M6-2F9; M6-1D4; M6-1F3; M6-1B9; and M6-1E9. Biochemical characterizations and cross-blocking experiments indicated that M6-1B9 and M6-1E9 recognize the same or contiguous epitopes on CD147. By employing COS transfectants expressing CD147 membrane-distal domain (domain 1) and membrane-proximal domain (domain 2), mAbs M6-2F9, M6-1D4, M6-1B9, and M6-1E9 were shown to recognize epitopes located on domain 1 of the molecule. Functional studies indicated that engagement of CD147 by mAbs M6-1B9 and M6-1E9 strongly inhibited lymphocyte proliferation induced by a CD3 mAb. In contrast, mAbs M6-2F9, M6-1D4, and M6-1F3 induced U937 homotypic cell aggregation. The results indicate that CD147 contains at least two bioactive domains. Epitopes responsible for induction of cell aggregation are different from those regulating lymphocyte activation.

Adhesion inhibition of Mycoplasma iowae to chicken lymphoma DT40 cells by monoclonal antibodies reacting with a 65-kD polypeptide.

PubMed

Fiorentin, L; Panangala, V S; Zhang, Y; Toivio-Kinnucan, M

1998-01-01

Tissue- and cell-specific attachment of mycoplasmas is a key aspect of the host-parasite relationship. In this study, monoclonal antibodies (MAbs) recognizing surface membrane polypeptides with molecular masses of 46 kD (p46) and 65 kD (p65), respectively, were examined in a microtiter cell attachment (agglutination) inhibition assay. MAbs MI3, MI6, and MI12 reacting with p65 polypeptide of Mycoplasma iowae inhibited attachment of the organisms to chicken lymphoma (DT 40) cells. One MAb (MI2) that reacted with p65 in immunoblots did not inhibit cell attachment, possibly because of the intrinsic native conformation of the epitope(s) in intact mycoplasmas as opposed to the linear state (sodium dodecyl sulfate denatured) in immunoblots. More pronounced M. iowae adherence inhibition was demonstrated by polyclonal turkey and mouse anti-M. iowae antisera compared with MAbs. Immunogold labelling followed by electron microscopy allowed us to localize the MAb-recognized epitopes on the membrane surface of M. iowae. On the basis of the cell attachment inhibition of M. iowae by specific MAbs (MI3, MI6, and MI12), we propose that the p65 polypeptide plays a role in cytadherence. The ability of polyclonal antisera to inhibit attachment of M. iowae more efficiently than the MAbs suggests that additional epitopes within p65 and/or other proteins are involved in cell attachment.

Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype.

PubMed

Hillen, Nina; Mester, Gabor; Lemmel, Claudia; Weinzierl, Andreas O; Müller, Margret; Wernet, Dorothee; Hennenlotter, Jörg; Stenzl, Arnulf; Rammensee, Hans-Georg; Stevanović, Stefan

2008-11-01

Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide-based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA-B44 supertype (HLA-B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule. Further direct comparisons by two mass spectrometric methods--isotope labeling as well as a label-free approach--consistently demonstrated only minute overlaps of below 3% between the ligandomes of different HLA antigens. In addition, T cell reactions of healthy donors against immunodominant HLA-B*44 and HLA-B*40 epitopes from EBV lacked promiscuous T-cell recognition within the HLA-B44 supertype. Taken together, these results challenge the common paradigm of broadly presented epitopes within this supertype.

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences

PubMed Central

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D.; Georgiev, Ivelin S.

2014-01-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

The grafting of universal T-helper epitopes enhances immunogenicity of HIV-1 Tat concurrently improving its safety profile.

PubMed

Kashi, Venkatesh P; Jacob, Rajesh A; Shamanna, Raghavendra A; Menon, Malini; Balasiddaiah, Anangi; Varghese, Rebu K; Bachu, Mahesh; Ranga, Udaykumar

2014-01-01

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity.

PubMed

Däumer, Martin P; Schneider, Beate; Giesen, Doris M; Aziz, Sheriff; Kaiser, Rolf; Kupfer, Bernd; Schneweis, Karl E; Schneider-Mergener, Jens; Reineke, Ulrich; Matz, Bertfried; Eis-Hübinger, Anna M

2011-05-01

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

Diagnostic Peptide Discovery: Prioritization of Pathogen Diagnostic Markers Using Multiple Features

PubMed Central

Carmona, Santiago J.; Sartor, Paula A.; Leguizamón, María S.; Campetella, Oscar E.; Agüero, Fernán

2012-01-01

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete “peptidomes”. Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked 10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes. PMID:23272069

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

PubMed

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Kurosawa, Yoshikazu; Yokoyama, Shigeyuki

2014-07-01

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

PubMed Central

Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

2014-01-01

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines. PMID:24719430

Analysis of Individuals from a Dengue-Endemic Region Helps Define the Footprint and Repertoire of Antibodies Targeting Dengue Virus 3 Type-Specific Epitopes

PubMed Central

Andrade, Daniela V.; Katzelnick, Leah C.; Widman, Doug G.; Balmaseda, Angel; de Silva, Aravinda M.; Baric, Ralph S.

2017-01-01

ABSTRACT The four dengue virus serotypes (DENV1 to 4) cause dengue, a major public health problem worldwide. Individuals exposed to primary DENV infections develop serotype-specific neutralizing antibodies, including strongly neutralizing antibodies targeting quaternary epitopes. To date, no studies have measured the levels and kinetics of serum antibodies directed to such epitopes among populations in regions where dengue is endemic. Here, we use a recombinant DENV4 (rDENV4/3-M14) displaying a major DENV3 type-specific quaternary epitope recognized by human monoclonal antibody 5J7 to measure the proportion, magnitude, and kinetics of DENV3 type-specific neutralizing antibody responses targeting this epitope. Primary DENV3 sera from 30 individuals in a dengue hospital-based study in Nicaragua were studied 3, 6, 12, and 18 months post-infection, alongside samples collected annually 1 to 4 years post-primary DENV3 infection from 10 individuals in a cohort study in Nicaragua. We found substantial individual variation in the proportion of DENV3 type-specific neutralizing antibody titers attributed to the 5J7 epitope (range, 0 to 100%), with the mean significantly increasing from 22.6% to 41.4% from 3 to 18 months. We extended the transplanted DENV3 5J7 epitope on the virion (rDENV4/3-M16), resulting in increased recognition in several individuals, helping define the footprint of the epitope. However, 37% and 13% of the subjects still showed little to no recognition of the 5J7 epitope at 3 and 18 months, respectively, indicating that one or more additional DENV3 type-specific epitopes exist. Overall, this study demonstrates how DENV-immune plasma from populations from areas of endemicity, when coupled with structurally guided recombinant viruses, can help characterize the epitope-specific neutralizing antibody response in natural DENV infections, with direct implications for design and evaluation of dengue vaccines. PMID:28928210

In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations

PubMed Central

Yang, Yi; Sun, Weilai; Guo, Jingjing; Zhao, Guangyu; Sun, Shihui; Yu, Hong; Guo, Yan; Li, Jungfeng; Jin, Xia; Du, Lanying; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

2015-01-01

The development of an HIV-1 vaccine that is capable of inducing effective and broadly cross-reactive humoral and cellular immune responses remains a challenging task because of the extensive diversity of HIV-1, the difference of virus subtypes (clades) in different geographical regions, and the polymorphism of human leukocyte antigens (HLA). We performed an in silico design of 3 DNA vaccines, designated pJW4303-MEG1, pJW4303-MEG2 and pJW4303-MEG3, encoding multi-epitopes that are highly conserved within the HIV-1 subtypes most prevalent in China and can be recognized through HLA alleles dominant in China. The pJW4303-MEG1-encoded protein consisted of one Th epitope in Env, and one, 2, and 6 epitopes in Pol, Env, and Gag proteins, respectively, with a GGGS linker sequence between epitopes. The pJW4303-MEG2-encoded protein contained similar epitopes in a different order, but with the same linker as pJW4303-MEG1. The pJW4303-MEG3-encoded protein contained the same epitopes in the same order as that of pJW4303-MEG2, but with a different linker sequence (AAY). To evaluate immunogenicity, mice were immunized intramuscularly with these DNA vaccines. Both pJW4303-MEG1 and pJW4303-MEG2 vaccines induced equally potent humoral and cellular immune responses in the vaccinated mice, while pJW4303-MEG3 did not induce immune responses. These results indicate that both epitope and linker sequences are important in designing effective epitope-based vaccines against HIV-1 and other viruses. PMID:25839222

Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

PubMed

Berger, Peter; Lapthorn, Adrian J

2016-01-01

hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

PubMed Central

Dey, Antu K.; Burke, Brian; Sun, Yide; Sirokman, Klara; Nandi, Avishek; Hartog, Karin; Lian, Ying; Geonnotti, Anthony R.; Montefiori, David; Franti, Michael; Martin, Grégoire; Carfi, Andrea; Kessler, Pascal; Martin, Loïc; Srivastava, Indresh K.; Barnett, Susan W.

2012-01-01

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved “CD4 induced” (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-27312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application. PMID:22291921

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs

PubMed Central

Brooks, Benjamin D.; Friedman, Harvey M.

2018-01-01

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen. PMID:29791513

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

PubMed

Hook, Lauren M; Cairns, Tina M; Awasthi, Sita; Brooks, Benjamin D; Ditto, Noah T; Eisenberg, Roselyn J; Cohen, Gary H; Friedman, Harvey M

2018-05-01

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen.

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts

PubMed Central

Archila, Luis Diego; Chow, I-Ting; McGinty, John W.; Renand, Amedee; Jeong, David; Robinson, David; Farrington, Mary L.; Kwok, William.W.

2017-01-01

Background Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. Objectives In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. Methods CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. Results CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH2 or TH2/TH17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Conclusions Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH2 type response at an epitope specific level. Clinical relevance Lack of cross-reactivity between walnut and cashew suggest that cashew peptide immunotherapy approach may not be most effective for walnut. PMID:27129138

Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

PubMed

Archila, L D; Chow, I-T; McGinty, J W; Renand, A; Jeong, D; Robinson, D; Farrington, M L; Kwok, W W

2016-06-01

Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut. © 2016 John Wiley & Sons Ltd.

CD18 activation epitopes induced by leukocyte activation.

PubMed

Beals, C R; Edwards, A C; Gottschalk, R J; Kuijpers, T W; Staunton, D E

2001-12-01

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

Amino acid residues 196-225 of LcrV represent a plague protective epitope.

PubMed

Quenee, Lauriane E; Berube, Bryan J; Segal, Joshua; Elli, Derek; Ciletti, Nancy A; Anderson, Deborah; Schneewind, Olaf

2010-02-17

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196-225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196-225 retained the ability of eliciting plague protection. These results identify LcrV residues 196-225 as a linear epitope that is recognized by the murine immune system to confer plague protection. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Amino acid residues 196–225 of LcrV represent a plague protective epitope

PubMed Central

Quenee, Lauriane E.; Berube, Bryan J.; Segal, Joshua; Elli, Derek; Ciletti, Nancy A.; Anderson, Deborah; Schneewind, Olaf

2010-01-01

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196–225 were necessary and sufficient for MAb-BA5 binding. Compared to full length LcrV, a variant lacking its residues 196–225 retained the ability of eliciting plague protection. These results identify LcrV residues 196–225 as a linear epitope that is recognized by the murine immune system to confer plague protection. PMID:20005318

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

PubMed

Hilton, H G; Parham, P

2013-04-01

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

HIV Molecular Immunology 2015

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yusim, Karina; Korber, Bette Tina; Brander, Christian

The scope and purpose of the HIV molecular immunology database: HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2015 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/ content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and bindingmore » sites within each protein, moving from left to right through the coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as cross-reactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins are provided. Alignments of CTL, helper T-cell, and antibody epitopes are available through the search interface on our web site at http:// www.hiv.lanl.gov/content/immunology.« less

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus.

PubMed

Okano, M; Nagano, T; Nakada, M; Masuda, Y; Kino, K; Yasueda, H; Nose, Y; Nishimura, Y; Ohta, N

1996-01-01

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus, were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and I58-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1*1502, and the latter by HLA-DRB1*0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA- DRB1*1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1*1502. The epitope peptide was also recognized by HLA-DRB1*1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.

Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran.

PubMed

Zhand, Sareh; Tabarraei, Alijan; Nazari, Amineh; Moradi, Abdolvahab

2017-07-01

Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran. In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559). The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes' (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions. CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21.

PubMed

Leung, Isabel; Dekel, Ayelet; Shifman, Julia M; Sidhu, Sachdev S

2016-08-02

A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.

Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

PubMed

Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

2016-05-11

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

Identification of an immunodominant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

PubMed

Rodriguez, M J; Sarraseca, J; Fominaya, J; Cortés, E; Sanz, A; Casal, J I

2001-05-01

Glycoprotein 5 (GP(5)) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). Expression of GP(5) has been improved by removing the transmembrane regions. Vectors were constructed encoding complete GP(5) plus three mutants: GP(5) Ns (residues 28--201), GP(5)[30--67] (residues 30--67) and GP(5)[30--201] (residues 30--67/130--201). The three deletion mutants were expressed at levels 20--30 times higher than complete GP(5). GP(5)[30--201] was well recognized in ELISA or immunoblotting by a collection of pig sera. All the fragments were tested for the generation of MAbs, but only the polyhistidine-tagged fragment GP(5)[30--201]H elicited an antibody response sufficient to produce MABS: The two MAbs were positive for PRRSV in ELISA and immunoblotting, but negative for virus neutralization. MAb 4BE12 reacted with residues 130--170 and MAb 3AH9 recognized residues 170--201. This region was recognized strongly in immunoblotting by a collection of infected-pig sera. These results indicate diagnostic potential for this epitope.

Carbohydrates and T cells: A sweet twosome

PubMed Central

Avci, Fikri Y.; Li, Xiangming; Tsuji, Moriya; Kasper, Dennis L.

2013-01-01

Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically-linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease. PMID:23757291

Fragments of the V1/V2 domain of HIV-1 glycoprotein 120 engineered for improved binding to the broadly neutralizing PG9 antibody.

PubMed

Morales, Javier F; Yu, Bin; Perez, Gerardo; Mesa, Kathryn A; Alexander, David L; Berman, Phillip W

2016-09-01

The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

PubMed Central

Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

2009-01-01

Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584

Inability to induce consistent T-cell responses recognizing conserved regions within HIIV-1 antigens: a potential mechanism for lack of vaccine efficacy in the step study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korber, Bette; Szinger, James

2009-01-01

T cell based vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a high probability of matching the epitope induced by vaccination with the infecting viral strain. We compared the frequency and specificity of the CTL epitopes elicited by the replication defective AdS gag/pol/nef vaccine used in the STEP trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. On average vaccination elicited only one epitope per gene. Importantly, the highly conservedmore » epitopes in gag, pol, and nef (> 80% of strains in the current collection of the Los Alamos database [www.hiv.lanl.gov]) were rarely elicited by vaccination. Moreover there was a statistically significant skewing of the T cell response to relative variable epitopes of each gene; only 20% of persons possessed > 3 T cell responses to epitopes likely to be found in circulating strains in the CladeB populations in which the Step trial was conducted. This inability to elicit T cell responses likely to be found in circulating viral strains is a likely factor in the lack of efficacy of the vaccine utilized in the STEP trial. Modeling of the epitope specific responses elicited by vaccination, we project that a median of 8-10 CD8+ T cell epitopes are required to provide >80% likelihood of eliciting at least 3 CD8+ T cell epitopes that would be found on a circulating population of viruses. Development of vaccine regimens which elicit either a greater breadth of responses or elicit responses to conserved regions of the HIV-1 genome are needed to fully evaluate the concept of whether induction of T cell immunity can alter HIV-1 in vivo.« less

Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

NASA Astrophysics Data System (ADS)

Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

1987-05-01

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

A zinc-dependent epitope on the molecule of thymulin, a thymic hormone.

PubMed Central

Dardenne, M; Savino, W; Berrih, S; Bach, J F

1985-01-01

Thymulin is a nonapeptide hormone produced by thymic epithelial cells. Its biological activity is strictly dependent on the presence of the metal zinc in the molecule. Antithymulin monoclonal antibodies have been produced against either the synthetic (AS1) or the natural intraepithelial (AE1) molecule. These monoclonal antibodies were screened for their abilities to inhibit the zinc-dependent biological activity of the hormone and were shown to bind to thymic epithelial cells. By using biological and immunofluorescence assays, the two antibodies were shown to recognize exclusively the zinc-coupled thymulin molecule. Other antithymulin antibodies screened by RIA or ELISA (using a zinc-deprived substrate) recognized a zinc-independent epitope on the thymulin molecule. These data indicate the existence of a zinc-specific conformation on the thymulin molecule. They are in agreement with NMR studies showing that the zinc-containing hormone has a unique structure. Images PMID:2413455

Epitope mapping of anti-interleukin-13 neutralizing antibody CNTO607.

PubMed

Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L

2009-05-29

CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

Fine Specificity Mapping of Autoantigens Targeted by Anti-Centromere Autoantibodies

PubMed Central

Akbarali, Yasmin; Matousek-Ronck, Jennifer; Hunt, Laura; Staudt, Leslie; Reichlin, Morris; Guthridge, Joel M.; James, Judith A

2007-01-01

Summary Autoantibodies to centromeric proteins are commonly found in sera of limited scleroderma and other rheumatic disease patients. To better understand the inciting events and possible pathogenic mechanisms of these autoimmune responses, this study identified the common antigenic targets of CENP-A in scleroderma patient sera. Utilizing samples from 263 anti-centromere immunofluorescence positive patients, 93.5% were found to have anti-CENP-A reactivity and 95.4% had anti-CENP-B reactivity by ELISA. Very few patient samples exclusively targeted CENP-A (2.7%) or CENP-B (4.2%). Select patient sera were tested for reactivity with solid phase overlapping decapeptides of CENP-A. Four distinct epitopes of CENP-A were identified. Epitopes 2 and 3 were confirmed by additional testing of 263 patient sera by ELISA for reactivity with these sequences constructed as multiple antigenic peptides. Inhibition CENP-A Western blots also confirmed the specificity of these humoral peptide immune responses in a subset of patient sera. The first three arginine residues (aa 4-6) of CENP-A appear essential for antibody recognition, as replacing these arginines with glycine residues reduced antibody binding to the expressed CENP-A protein by an average of 93.2% (range 80-100%). In selected patients with serial samples spanning nearly a decade, humoral epitope binding patterns were quite stable and showed no epitope spreading over time. This epitope mapping study identifies key antigenic targets of the anti-centromere response and establishes that the majority of the responses depend on key amino-terminal residues. PMID:17210244

Antibodies causing thrombocytopenia in patients treated with RGD-mimetic platelet inhibitors recognize ligand-specific conformers of αIIb/β3 integrin

PubMed Central

Rasmussen, Mark; Zhu, Jieqing; Aster, Richard H.

2012-01-01

Arginine-glycine-aspartic acid (RGD)–mimetic platelet inhibitors act by occupying the RGD recognition site of αIIb/β3 integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen. Thrombocytopenia is a well-known side effect of treatment with this class of drugs and is caused by Abs, often naturally occurring, that recognize αIIb/β3 in a complex with the drug being administered. RGD peptide and RGD-mimetic drugs are known to induce epitopes (ligand-induced binding sites [LIBS]) in αIIb/β3 that are recognized by certain mAbs. It has been speculated, but not shown experimentally, that Abs from patients who develop thrombocytopenia when treated with an RGD-mimetic inhibitor similarly recognize LIBS determinants. We addressed this question by comparing the reactions of patient Abs and LIBS-specific mAbs against αIIb/β3 in a complex with RGD and RGD-mimetic drugs, and by examining the ability of selected non-LIBS mAbs to block binding of patient Abs to the liganded integrin. Findings made provide evidence that the patient Abs recognize subtle, drug-induced structural changes in the integrin head region that are clustered about the RGD recognition site. The target epitopes differ from classic LIBS determinants, however, both in their location and by virtue of being largely drug-specific. PMID:22490676

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

PubMed Central

Carlson, Jonathan; Yan, Jiyu; Akinsiku, Olusimidele T.; Schaefer, Malinda; Sabbaj, Steffanie; Bet, Anne; Levy, David N.; Heath, Sonya; Tang, Jianming; Kaslow, Richard A.; Walker, Bruce D.; Ndung’u, Thumbi; Goulder, Philip J.; Heckerman, David; Hunter, Eric; Goepfert, Paul A.

2010-01-01

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity. PMID:20065064

Long-lived CD8+ T cell responses following Crimean-Congo haemorrhagic fever virus infection

PubMed Central

Goedhals, Dominique; Paweska, Janusz T.

2017-01-01

Crimean-Congo haemorrhagic fever virus (CCHFV) is a member of the Orthonairovirus genus of the Nairoviridae family and is associated with haemorrhagic fever in humans. Although T lymphocyte responses are known to play a role in protection from and clearance of viral infections, specific T cell epitopes have yet to be identified for CCHFV following infection. A panel of overlapping peptides covering the CCHFV nucleoprotein and the structural glycoproteins, GN and GC, were screened by ELISpot assay to detect interferon gamma (IFN-γ) production in vitro by peripheral blood mononuclear cells from eleven survivors with previous laboratory confirmed CCHFV infection. Reactive peptides were located predominantly on the nucleoprotein, with only one survivor reacting to two peptides from the glycoprotein GC. No single epitope was immunodominant, however all but one survivor showed reactivity to at least one T cell epitope. The responses were present at high frequency and detectable several years after the acute infection despite the absence of continued antigenic stimulation. T cell depletion studies confirmed that IFN-γ production as detected using the ELISpot assay was mediated chiefly by CD8+ T cells. This is the first description of CD8+ T cell epitopic regions for CCHFV and provides confirmation of long-lived T cell responses in survivors of CCHFV infection. PMID:29261651

Mechanisms of escape from the PGT128 family of anti-HIV broadly neutralizing antibodies.

PubMed

Krumm, Stefanie A; Mohammed, Hajer; Le, Khoa M; Crispin, Max; Wrin, Terri; Poignard, Pascal; Burton, Dennis R; Doores, Katie J

2016-02-02

Broadly neutralizing antibodies (bnAbs) directed against the mannose-patch on the HIV envelope glycoprotein gp120 have several features that make them desirable targets for vaccine design. The PGT125-131 bnAb family is of particular interest due to its superior breadth and potency. The overlapping epitopes recognized by this family are intricate and neutralization requires interaction with at least two N-linked glycans (N332/N334, N295 or N301) in addition to backbone-mediated contact with the (323)IGDIR(327) motif of the V3 loop. We have recently shown that this bnAb family consists of two distinct antibody classes that can bind alternate arrangements of glycans in the mannose-patch in the absence of N332 thereby limiting viral escape. This led us to further investigate viral resistance and escape mechanisms to the PGT125-131 bnAb family. Using an escape virus isolated from the PGT125-131 donor as a guide, we show that mutating both the V3 core protein epitope and repositioning critical N-linked glycosylation sites are required to restore neutralization sensitivity. Interestingly, neutralization sensitivity could be restored via different routes for the two distinct bnAb classes within the PGT125-131 family, which may have been important in generating the divergence in recognition. We demonstrate that the observed V3 mutations confer neutralization resistance in other virus strains through both gain-of-function and escape studies. Furthermore, we show that the V3 loop is important in facilitating promiscuous binding to glycans within the mannose-patch. These data highlight the importance of the V3 loop in the design of immunogens aimed at inducing broad and potent bnAbs that can bind promiscuously to the mannose-patch.

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

PubMed

Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Repeat region of Brugia malayi sheath protein (Shp-1) carries Dominant B epitopes recognized in filarial endemic population.

PubMed

Jawaharlal, Jeya Prita Parasurama; Madhumathi, Jayaprakasam; Prince, Rajaiah Prabhu; Kaliraj, Perumal

2014-09-01

Transmission of lymphatic filariasis is mediated through microfilariae (L1 stage of the parasite) which is encased in an eggshell called sheath. The sheath protein Shp-1 stabilizes the structure due to the unique repeat region with Met-Pro-Pro-Gln-Gly sequences. Microfilarial proteins could be used as transmission blocking vaccines. Since the repeat region of Shp-1 was predicted to carry putative B epitopes, this region was used to analyze its reactivity with clinical samples towards construction of peptide vaccine. In silico analysis of Shp-1 showed the presence of B epitopes in the region 49-107. The polypeptide epitopic region Shp-149-107 was cloned and expressed in Escherichia coli. Antibody reactivity of the Shp-149-107 construct was evaluated in filarial endemic population by ELISA. Putatively immune endemic normals (EN) showed significantly high reactivity (P < 0.05) when compared to all the other categories. Antibody reactivity of Shp-1 repeat region was similar to that of whole protein proving that this region carries B epitopes responsible for its humoral response in humans. Thus this can be employed for inducing anti-microfilarial immunity in the infected population that may lead to reduction in transmission intensity and also it could be used along with other epitopes from different stages of the parasite in order to manage the disease effectively.

Activation of effector functions by immune complexes of mouse IgG2a with isotype-specific autoantibodies.

PubMed Central

Rajnavölgyi, E; Fazekas, G; Lund, J; Daeron, M; Teillaud, J L; Jefferis, R; Fridman, W H; Gergely, J

1995-01-01

Analysis of five monoclonal autoantibodies, rheumatoid factors produced by hybridomas generated from spleen cells of BALB/c mice repeatedly infected with A/PR/8/34 human influenza A virus, revealed that they recognized distinct but spatially related epitopes. The differing isoallotypic specificity of the IgM and IgA monoclonal antibodies correlated with the presence of Ile258 and Ala305, respectively. Although these data suggest that the epitopes recognized are within the CH2 domain, all antibodies failed to inhibit IgG antigen reactivity with Staphylococcus aureus protein A (SpA), C1q, mouse C3, human Fc gamma RI or mouse Fc gamma RII, activities known to be predominantly determined by CH2 domain structures. Reactivity of the IgA antibody, Z34, with IgG2b allowed further specificity studies using a panel of 26 mutant IgG2b proteins, each having single amino acid replacements over the surface of the CH2 domain. The only substitution that affected Z34 reactivity was Asn/Ala297, which destroyed the glycosylation sequon, resulting in secretion of an aglycosylated IgG molecule. The epitope recognized by Z34 therefore seems to be located outside of the Fc gamma R and C1q binding sites, but to be dependent on the presence of carbohydrate for expression. In contrast to the binding studies, complement activation by aggregated IgG2a, through classical or alternative pathways, was inhibited by the presence of autoantibodies. The functional significance of isotype-specific autoantibody in immune regulation is discussed. PMID:7540592

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

ANTI-11[E]-PYROGLUTAMATE-MODIFIED AMYLOID β ANTIBODIES CROSS-REACT WITH OTHER PATHOLOGICAL Aβ SPECIES: RELEVANCE FOR IMMUNOTHERAPY

PubMed Central

Perez-Garmendia, Roxanna; Ibarra-Bracamontes, Vanessa; Vasilevko, Vitaly; Luna-Muñoz, Jose; Mena, Raul; Govezensky, Tzipe; Acero, Gonzalo; Manoutcharian, Karen; Cribbs, David H.; Gevorkian, Goar

2010-01-01

N-truncated/modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as animal models of AD, and represent highly desirable therapeutic targets. In the present study we have focused on Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 11 (AβN11(pE)). We identified two B-cell epitopes recognized by rabbit anti-AβN11(pE) polyclonal antibodies. Interestingly, rabbit anti-AβN11(pE) polyclonal antibodies bound also to full-length Aβ1-42 and N-truncated/modified AβN3(pE), suggesting that the three peptides may share a common B-cell epitope. Importantly, rabbit anti-AβN11(pE) antibodies bound to naturally occurring Aβ aggregates present in brain samples from AD patients. These results are potentially important for developing novel immunogens for targeting N-truncated/modified Aβ aggregates as well, since the most commonly used immunogens in the majority of vaccine studies have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:20864186

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

PubMed Central

Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

2012-01-01

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

3-O-sulfated heparan sulfate recognized by the antibody HS4C3 contributes [corrected] to the differentiation of mouse embryonic stem cells via fas signaling.

PubMed

Hirano, Kazumi; Sasaki, Norihiko; Ichimiya, Tomomi; Miura, Taichi; Van Kuppevelt, Toin H; Nishihara, Shoko

2012-01-01

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.

3-O-Sulfated Heparan Sulfate Recognized by the Antibody HS4C3 Contribute to the Differentiation of Mouse Embryonic Stem Cells via Fas Signaling

PubMed Central

Hirano, Kazumi; Sasaki, Norihiko; Ichimiya, Tomomi; Miura, Taichi; Van Kuppevelt, Toin H.; Nishihara, Shoko

2012-01-01

Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs. PMID:22916262

In silico epitope prediction, expression and functional analysis of Per a 10 allergen from the American cockroach

PubMed Central

Tong, Xunliang; Guo, Miao; Jin, Min; Chen, Hao; Li, Yanming; Wei, Ji-Fu

2016-01-01

Cockroach (CR) allergies caused by the American cockroach hyave been recognized to be repsonsible for IgE-mediated type I hypersensitivity worldwide. Per a 10 is one of the recognized main allergens of the American CR. In a previous study, we examined another American CR allergen, Per a 9 in patients with CR allergies and examined epitope sequences in this allergen. In the present study, we aimed to examine epitope sequences in the Per a 10 allergen. for this purpose, the Per a 10 gene was cloned and expressed in Escherichia coli (E. coli) systems. Our results revealed that 9 out of 16 (56.3%) sera from patients with American CR allergies reacted to Per a10, as assessed by ELISA, confirming that Per a 10 is a major allergen of the American CR. Our results also revealed that the expression of CD63 and CCR3 on passively sensitized basophils (obtained sera of patients with American CR allergies) was increased by approximately 2.3-fold, indicating that recombinant Per a 10 is functionally active. In addition, 3 immunoinformatics tools, namely the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the peptides and the results revealed 8 peptides (2–12, 55–67, 98–120, 125–133, 149–160, 170–182, 201–208 and 223–227) as potential B cell epitopes of the Per a 10 allergen. Moreover, Per a 10 was predicted to have 3 T cell epitope sequences, namely 83–92, 139–147 and 162–170. The findings of our study on the CR allergen may prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of CR allergies. PMID:27840898

In silico epitope prediction, expression and functional analysis of Per a 10 allergen from the American cockroach.

PubMed

Tong, Xunliang; Guo, Miao; Jin, Min; Chen, Hao; Li, Yanming; Wei, Ji-Fu

2016-12-01

Cockroach (CR) allergies caused by the American cockroach hyave been recognized to be repsonsible for IgE-mediated type I hypersensitivity worldwide. Per a 10 is one of the recognized main allergens of the American CR. In a previous study, we examined another American CR allergen, Per a 9 in patients with CR allergies and examined epitope sequences in this allergen. In the present study, we aimed to examine epitope sequences in the Per a 10 allergen. for this purpose, the Per a 10 gene was cloned and expressed in Escherichia coli (E. coli) systems. Our results revealed that 9 out of 16 (56.3%) sera from patients with American CR allergies reacted to Per a10, as assessed by ELISA, confirming that Per a 10 is a major allergen of the American CR. Our results also revealed that the expression of CD63 and CCR3 on passively sensitized basophils (obtained sera of patients with American CR allergies) was increased by approximately 2.3-fold, indicating that recombinant Per a 10 is functionally active. In addition, 3 immunoinformatics tools, namely the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the peptides and the results revealed 8 peptides (2-12, 55-67, 98-120, 125-133, 149-160, 170-182, 201-208 and 223-227) as potential B cell epitopes of the Per a 10 allergen. Moreover, Per a 10 was predicted to have 3 T cell epitope sequences, namely 83-92, 139-147 and 162-170. The findings of our study on the CR allergen may prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of CR allergies.

Development and characterization of a panel of cross-reactive monoclonal antibodies generated using H1N1 influenza virus.

PubMed

Guo, Chun-yan; Tang, Yi-gui; Qi, Zong-li; Liu, Yang; Zhao, Xiang-rong; Huo, Xue-ping; Li, Yan; Feng, Qing; Zhao, Peng-hua; Wang, Xin; Li, Yuan; Wang, Hai-fang; Hu, Jun; Zhang, Xin-jian

2015-08-01

To characterize the antigenic epitopes of the hemagglutinin (HA) protein of H1N1 influenza virus, a panel consisting of 84 clones of murine monoclonal antibodies (mAbs) were generated using the HA proteins from the 2009 pandemic H1N1 vaccine lysate and the seasonal influenza H1N1(A1) vaccines. Thirty-three (39%) of the 84 mAbs were found to be strain-specific, and 6 (7%) of the 84 mAbs were subtype-specific. Twenty (24%) of the 84 mAbs recognized the common HA epitopes shared by 2009 pandemic H1N1, seasonal A1 (H1N1), and A3 (H3N2) influenza viruses. Twenty-five of the 84 clones recognized the common HA epitopes shared by the 2009 pandemic H1N1, seasonal A1 (H1N1) and A3 (H3N2) human influenza viruses, and H5N1 and H9N2 avian influenza viruses. We found that of the 16 (19%) clones of the 84 mAbs panel that were cross-reactive with human respiratory pathogens, 15 were made using the HA of the seasonal A1 (H1N1) virus and 1 was made using the HA of the 2009 pandemic H1N1 influenza virus. Immunohistochemical analysis of the tissue microarray (TMA) showed that 4 of the 84 mAb clones cross-reacted with human tissue (brain and pancreas). Our results indicated that the influenza virus HA antigenic epitopes not only induce type-, subtype-, and strain-specific monoclonal antibodies against influenza A virus but also cross-reactive monoclonal antibodies against human tissues. Further investigations of these cross-reactive (heterophilic) epitopes may significantly improve our understanding of viral antigenic variation, epidemics, pathophysiologic mechanisms, and adverse effects of influenza vaccines. Copyright © 2015 Elsevier GmbH. All rights reserved.

Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus

PubMed Central

Olal, Daniel; Kuehne, Ana I.; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L.; Lee, Jeffrey E.; King, Liam B.; Kawaoka, Yoshihiro; Dye, John M.

2012-01-01

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics. PMID:22171276

Structure of an antibody in complex with its mucin domain linear epitope that is protective against Ebola virus.

PubMed

Olal, Daniel; Kuehne, Ana I; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L; Lee, Jeffrey E; King, Liam B; Kawaoka, Yoshihiro; Dye, John M; Saphire, Erica Ollmann

2012-03-01

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.

Metal chelation dual-template epitope imprinting polymer via distillation-precipitation polymerization for recognition of porcine serum albumin.

PubMed

Qin, Ya-Ping; Wang, Hai-Yan; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui

2018-08-01

A novel dual-template epitope imprinting polymer coated on magnetic carbon nanotubes (MCNTs@D-EMIP) was successfully prepared for specific recognition of porcine serum albumin (PSA) via dual-template epitope imprinting, metal chelation imprinting and distillation-precipitation polymerization (DPP). C-terminal peptides and N-terminal peptides of PSA were selected as templates simultaneously, and zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were used as functional monomer and cross-linker, respectively. The epitope templates were immobilized by metal chelation and six-membered ring formed with zinc acrylate. Finally, MCNTs@D-EMIP was synthesized by DPP in only 30 min, which was much shorter than those of other polymerization methods. The prepared MCNTs@D-EMIP displayed specific recognition ability toward PSA and its adsorption amount and imprinting factor were 45.05 mg g -1 and 4.50, which were much higher than those of single template epitope imprinting polymers. Besides, high-performance liquid chromatography (HPLC) analysis of PSA in porcine blood serum real sample indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@D-EMIP had potential to be applied in bio-separation area. In addition, the results of cross-reactivity experiment proved that this strategy had generality to prepare dual-template epitope imprinting polymer for recognition of target protein. In summary, this study provided an efficient protocol to recognize target protein in complex sample via dual-template epitope imprinting approach, metal chelation imprinting and distillation-precipitation polymerization. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

PubMed

Chen, Yiyang; Liu, Baoyuan; Sun, Yani; Li, Huixia; Du, Taofeng; Nan, Yuchen; Hiscox, Julian A; Zhou, En-Min; Zhao, Qin

2018-07-01

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625 DFCP 628 , 458 PSRPF 462 , and 407 EPTV 410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design. IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts. Copyright © 2018 American Society for Microbiology.

Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague.

PubMed

Zvi, Anat; Rotem, Shahar; Zauberman, Ayelet; Elia, Uri; Aftalion, Moshe; Bar-Haim, Erez; Mamroud, Emanuelle; Cohen, Ofer

2017-10-20

The causative agent of Plague, Yersinia pestis, is a highly virulent pathogen and a potential bioweapon. Depending on the route of infection, two prevalent occurrences of the disease are known, bubonic and pneumonic. The latter has a high fatality rate. In the absence of a licensed vaccine, intense efforts to develop a safe and efficacious vaccine have been conducted, and humoral-driven subunit vaccines containing the F1 and LcrV antigens are currently under clinical trials. It is well known that a cellular immune response might have an essential additive value to immunity and protection against Y. pestis infection. Nevertheless, very few documented epitopes eliciting a protective T-cell response have been reported. Here, we present a combined high throughput computational and experimental effort towards identification of CD8 T-cell epitopes. All 4067 proteins of Y. pestis were analyzed with state-of-the-art recently developed prediction algorithms aimed at mapping potential MHC class I binders. A compilation of the results obtained from several prediction methods revealed a total of 238,000 peptide candidates, which necessitated downstream filtering criteria. Our previously established and proven approach for enrichment of true positive CTL epitopes, which relies on mapping clusters rich in tandem or overlapping predicted MHC binders ("hotspots"), was applied, as well as considerations of predicted binding affinity. A total of 1532 peptides were tested for their ability to elicit a specific T-cell response by following the production of IFNγ from splenocytes isolated from vaccinated mice. Altogether, the screen resulted in 178 positive responders (11.8%), all novel Y. pestis CTL epitopes. These epitopes span 113 Y. pestis proteins. Substantial enrichment of membrane-associated proteins was detected for epitopes selected from hotspots of predicted MHC binders. These results considerably expand the repertoire of known CTL epitopes in Y. pestis and pave the way to attest their protective potential, and hence their contribution to a future potent subunit vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice.

PubMed

Harrison, L C; Honeyman, M C; Trembleau, S; Gregori, S; Gallazzi, F; Augstein, P; Brusic, V; Hammer, J; Adorini, L

1997-03-17

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Development of Peptide Vaccines in Dengue.

PubMed

Reginald, Kavita; Chan, Yanqi; Plebanski, Magdalena; Poh, Chit Laa

2017-09-13

Dengue is one of the most important arboviral infection worldwide, infecting up to 390 million people and causing 25,000 deaths annually. Although a licensed dengue vaccine is available, it is not efficacious against dengue serotypes that infect people living in South East Asia, where dengue is an endemic disease. Hence, there is an urgent need to develop an efficient dengue vaccine for this region. Data from different clinical trials indicate that a successful dengue vaccine must elicit both neutralizing antibodies and cell mediated immunity. This can be achieved by designing a multi-epitope peptide vaccine comprising B, CD8+ and CD4+ T cell epitopes. As recognition of T cell epitopes are restricted by human leukocyte antigens (HLA), T cell epitopes which are able to recognize several major HLAs will be preferentially included in the vaccine design. While peptide vaccines are safe, biocompatible and cost-effective, it is poorly immunogenic. Strategies to improve its immunogenicity by the use of long peptides, adjuvants and nanoparticle delivery mechanisms are discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8+ T cell epitopes

PubMed Central

Hammond, A S; Klein, M R; Corrah, T; Fox, A; Jaye, A; McAdam, K P; Brookes, R H

2005-01-01

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8+ T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-γ. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8+ T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8+ T cell responses appear to be widespread throughout the genome. PMID:15762882

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

PubMed

Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

2014-05-15

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

Towards Identifying Protective B-Cell Epitopes: The PspA Story.

PubMed

Khan, Naeem; Jan, Arif T

2017-01-01

Pneumococcal surface protein A (PspA) is one of the most abundant cell surface protein of Streptococcus pneumoniae ( S. pneumoniae ). PspA variants are structurally and serologically diverse and help evade complement-mediated phagocytosis of S. pneumoniae , which is essential for its survival in the host. PspA is currently been screened for employment in the generation of more effective (serotype independent) vaccine to overcome the limitations of polysaccharide based vaccines, providing serotype specific immune responses. The cross-protection eliciting regions of PspA localize to the α-helical and proline rich regions. Recent data indicate significant variation in the ability of antibodies induced against the recombinant PspA variants to recognize distinct S. pneumoniae strains. Hence, screening for the identification of the topographical repertoire of B-cell epitopes that elicit cross-protective immune response seems essential in the engineering of a superior PspA-based vaccine. Herein, we revisit epitope identification in PspA and the utility of hybridoma technology in directing the identification of protective epitope regions of PspA that can be used in vaccine research.

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αβT Cell Receptor Signatures.

PubMed

Grant, Emma J; Josephs, Tracy M; Valkenburg, Sophie A; Wooldridge, Linda; Hellard, Margaret; Rossjohn, Jamie; Bharadwaj, Mandvi; Kedzierska, Katherine; Gras, Stephanie

2016-11-18

αβT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M1 58 , respectively) or with the hepatitis C and influenza viruses (NS3 1073 and NA 231 , respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αβTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αβTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Immune Escape Mutations Detected within HIV-1 Epitopes Associated with Viral Control During Treatment Interruption

PubMed Central

Schweighardt, Becky; Wrin, Terri; Meiklejohn, Duncan A.; Spotts, Gerald; Petropoulos, Christos J.; Nixon, Douglas F.; Hecht, Frederick M.

2010-01-01

We analyzed immune responses in chronically HIV-infected individuals who took part in a treatment interruption (TI) trial designed for patients who initiated anti-retroviral therapy within 6 months of seroconversion. In the two subjects that exhibited the best viral control, we detected CD8+ T cell responses against 1-2 Gag epitopes during the early weeks of TI and a subsequent increase in the number of epitopes recognized by the later time points. Each of these subjects developed mutations within the epitopes targeted by the highest magnitude responses. In the subject with the worst viral control, we detected responses against two Gag epitopes throughout the entire TI and no Gag mutations. The magnitude of these responses increased dramatically with time, greatly exceeding those detected in the virologic controllers. The highest levels of contemporaneous autologous neutralizing antibody activity were detected in the virologic controllers, and a subsequent escape mutation developed within the envelope gene of one controller that abrogated the response. These data suggest that immune escape mutations are a sign of viral control during TI, and that the absence of immune escape mutations in the presence of high-levels of viral replication indicates the lack of an effective host immune response. PMID:19910798

Sugar epitopes as potential universal disease transmission blocking targets.

PubMed

Dinglasan, Rhoel R; Valenzuela, Jesús G; Azad, Abdu F

2005-01-01

One promising method to prevent vector-borne diseases is through the use of transmission blocking vaccines (TBVs). However, developing several anti-pathogen TBVs may be impractical. In this study, we have identified a conserved candidate carbohydrate target in the midguts of several Arthropod vectors. A screen of the novel GlycoChip glycan array found that the anti-carbohydrate malaria transmission blocking monoclonal antibody (MG96) preferentially recognized D-mannose (alpha) and the type II lactosamine disaccharide. The specificity for D-mannose was confirmed by competition ELISA using alpha-methyl mannoside as inhibitor. Con A, which identifies terminal mannose residues, did not inhibit MG96 reactivity with mosquito midgut lysates, suggesting that Con A has differential recognition of this monosaccharide. However, the jack bean lectin, Jacalin, which recognizes D-mannose (alpha), d-galactose (alpha/beta) and the T antigen, not only displays a similar banding profile to that recognized by MG96 on immunoblot but was also shown to effectively inhibit MG96. Wheat-germ agglutinin, which recognizes N-acetyllactosamine units, only partially inhibited MG96 reactivity. This highlights the contribution of both glycan moieties to the MG96 epitope or glycotope. Enzyme deglycosylation results suggest that MG96 recognizes a mannose alpha1-6 substitution on an O-linked oligosaccharide. Taken together, the data suggest that MG96 recognizes a discontinuous glycotope composed of Manalpha1-6 proximal to Galbeta1-4GlcNAc-alpha-O-R glycans on arthropod vector midguts. As such, these glycotopes may represent potential transmission blocking vaccine targets for a wide range of vector-borne pathogens.

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences.

PubMed

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D; Georgiev, Ivelin S

2014-07-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Preparation of High-Efficiency Cytochrome c-Imprinted Polymer on the Surface of Magnetic Carbon Nanotubes by Epitope Approach via Metal Chelation and Six-Membered Ring.

PubMed

Qin, Ya-Ping; Li, Dong-Yan; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui

2016-04-27

A novel epitope molecularly imprinted polymer on the surface of magnetic carbon nanotubes (MCNTs@EMIP) was successfully fabricated to specifically recognize target protein cytochrome c (Cyt C) with high performance. The peptides sequences corresponding to the surface-exposed C-terminus domains of Cyt C was selected as epitope template molecule, and commercially available zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were employed as functional monomer and cross-linker, respectively, to synthesize MIP via free radical polymerization. The epitope was immobilized via metal chelation and six-membered ring formed between the functional monomer and the hydroxyl and amino groups of the epitope. The resulting MCNTs@EMIP exhibited specific recognition ability toward target Cyt C including more satisfactory imprinting factor (about 11.7) than that of other reported imprinting methods. In addition, the MCNTs@EMIP demonstrated a high adsorption amount (about 780.0 mg g(-1)) and excellent selectivity. Besides, the magnetic property of the support material made the processes easy and highly efficient by assistance of an external magnetic field. High-performance liquid chromatography analysis of Cyt C in bovine blood real sample and protein mixture indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@EMIP had potential to be applied in bioseparation area. In brief, this study provided a new protocol to detect target protein in complex sample via epitope imprinting approach and surface imprinting strategy.

RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.

PubMed

Hosono-Fukao, Tomomi; Ohtake-Niimi, Shiori; Nishitsuji, Kazuchika; Hossain, Md Motarab; van Kuppevelt, Toin H; Michikawa, Makoto; Uchimura, Kenji

2011-11-01

RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and cytokines in central nervous tissues. Chemically modified heparins that lack the trisulfated disaccharides failed to inhibit the RB4CD12 recognition of HS chains. To determine the localization of the RB4CD12 anti-HS epitope in the brain, we performed an immunohistochemical analysis for cryocut sections of mouse brain. The RB4CD12 staining signals were colocalized with laminin and were detected abundantly in the vascular basement membrane. Bacterial heparinases eliminated the RB4CD12 staining signals. The RB4CD12 epitope localization was confirmed by immunoelectron microscopy. Western blotting analysis revealed that the size of a major RB4CD12-positive molecule is ∼460 kDa in a vessel-enriched fraction of the mouse brain. Disaccharide analysis with reversed-phase ion-pair HPLC showed that [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-] trisulfated disaccharide residues are present in HS purified from the vessel-enriched brain fraction. These results indicated that the RB4CD12 anti-HS epitope exists in large quantities in the brain vascular basement membrane. Copyright © 2011 Wiley-Liss, Inc.

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

PubMed

Rueda, P; Hurtado, A; del Barrio, M; Martínez-Torrecuadrada, J L; Kamstrup, S; Leclerc, C; Casal, J I

1999-10-10

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses. Copyright 1999 Academic Press.

Protective Efficacy of Serially Up-Ranked Subdominant CD8+ T Cell Epitopes against Virus Challenges

PubMed Central

Roshorm, Yaowaluck; Bridgeman, Anne; Létourneau, Sven; Liljeström, Peter; Potash, Mary Jane; Volsky, David J.; McMichael, Andrew J.; Hanke, Tomáš

2011-01-01

Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans. PMID:21625575

Myasthenia Gravis and the Tops and Bottoms of AChRs Antigenic Structure of the MIR and Specific Immunosuppression of EAMG Using AChR Cytoplasmic Domains

PubMed Central

Lindstrom, Jon; Luo, Jie; Kuryatov, Alexander

2009-01-01

The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of α1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR α3 subunits, but MG patient antibodies do not. By making chimeras of α1 subunits with α7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of α1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in α1/α7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human α1, β1, γ, δ, and ε subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG. PMID:18567851

Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.

PubMed

Yang, Xiaolan; Hu, Xiaolei; Xu, Bangtian; Wang, Xin; Qin, Jialin; He, Chenxiong; Xie, Yanling; Li, Yuanli; Liu, Lin; Liao, Fei

2014-06-17

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

PubMed Central

Weekes, Michael P.; Wills, Mark R.; Mynard, Kim; Carmichael, Andrew J.; Sissons, J. G. Patrick

1999-01-01

Human cytomegalovirus (HCMV)-specific CD8+ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo. PMID:9971792

Comparison of the Specificities of IgG, IgG-Subclass, IgA and IgM Reactivities in African and European HIV-Infected Individuals with an HIV-1 Clade C Proteome-Based Array

PubMed Central

Gallerano, Daniela; Ndlovu, Portia; Makupe, Ian; Focke-Tejkl, Margarete; Fauland, Kerstin; Wollmann, Eva; Puchhammer-Stöckl, Elisabeth; Keller, Walter; Sibanda, Elopy; Valenta, Rudolf

2015-01-01

A comprehensive set of recombinant proteins and peptides of the proteome of HIV-1 clade C was prepared and purified and used to measure IgG, IgG-subclass, IgA and IgM responses in HIV-infected patients from Sub-Saharan Africa, where clade C is predominant. As a comparison group, HIV-infected patients from Europe were tested. African and European patients showed an almost identical antibody reactivity profile in terms of epitope specificity and involvement of IgG, IgG subclass, IgA and IgM responses. A V3-peptide of gp120 was identified as major epitope recognized by IgG1>IgG2 = IgG4>IgG3, IgA>IgM antibodies and a C-terminal peptide represented another major peptide epitope for the four IgG subclasses. By contrast, gp41-derived-peptides were mainly recognized by IgG1 but not by the other IgG subclasses, IgA or IgM. Among the non-surface proteins, protease, reverse transcriptase+RNAseH, integrase, as well as the capsid and matrix proteins were the most frequently and strongly recognized antigens which showed broad IgG subclass and IgA reactivity. Specificities and magnitudes of antibody responses in African patients were stable during disease and antiretroviral treatment, and persisted despite severe T cell loss. Using a comprehensive panel of gp120, gp41 peptides and recombinant non-surface proteins of HIV-1 clade C we found an almost identical antibody recognition profile in African and European patients regarding epitopes and involved IgG-sublass, IgA- and IgM-responses. Immune recognition of gp120 peptides and non-surface proteins involved all four IgG subclasses and was indicative of a mixed Th1/Th2 immune response. The HIV-1 clade C proteome-based test allowed diagnosis and monitoring of antibody responses in the course of HIV-infections and assessment of isotype and subclass responses. PMID:25658330

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

PubMed

Messer, William B; Yount, Boyd L; Royal, Scott R; de Alwis, Ruklanthi; Widman, Douglas G; Smith, Scott A; Crowe, James E; Pfaff, Jennifer M; Kahle, Kristen M; Doranz, Benjamin J; Ibarra, Kristie D; Harris, Eva; de Silva, Aravinda M; Baric, Ralph S

2016-05-15

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Total immunoglobulin G and IgG1 subclass levels specific for the MSP-1(19) of Plasmodium falciparum are different in individuals with either processing-inhibitory, blocking or neutral antibodies.

PubMed

Omosun, Y O; Adoro, S; Anumudu, C I; Odaibo, A; Holder, A A; Nwagwu, M; Nwuba, R I

2010-06-01

Some MSP-1(19) specific antibodies that inhibit merozoite invasion also inhibit the secondary processing of MSP-1. However the binding of these inhibitory antibodies can be blocked by another group of antibodies, called blocking antibodies, which recognize adjacent or overlapping epitopes, but themselves have no effect on either MSP-1 processing or merozoite invasion. These antibodies have been reported to be present in individuals living in a malaria endemic area. Blood samples were obtained from children shown to have processing inhibitory, blocking, and neutral antibodies in a previous study. Enzyme linked immunosorbent assay (ELISA), was used to determine the total IgG, IgM and IgG subtypes. There was a significant difference in anti-MSP-1(19) IgG, while there was no significant difference in the anti-MSP-1(19) IgM. Only anti MSP-1(19) IgG1, amongst the IgG subtypes was significantly different between the groups. This study shows that antibodies against MSP-1 are different not only in specificity and function but also in the amount of total IgG and IgG subtype produced.

The frequent mutation Gly/Asp in CDR1H may determine a cross-reactive idiotope in anti-I cold agglutinins

PubMed Central

ABATANGELO, C; PLOTKIN, L; MATHOV, I; SQUIQUERA, L; LEONI, J

1996-01-01

Variable domains (VH) of all known anti i/I cold agglutinin (CA) heavy chains are codified by the VH4–21 gene. While anti-i CAs are the expression of gene rearrangement without mutations represented by amino acid changes, anti-I CAs present, among others, a frequent somatic mutation of Gly by Asp at position 31. The hydropathy profile calculated for the CDR1H (position 30 to position 35), as well as some adjacent positions of the heavy chain belonging to anti-i and anti-I antibodies, showed the conformational changes accompanying the replacement of Gly by Asp. A MoAb (LP91), which had been obtained in BALB/c mice immunized with a Fabμ fragment from a monoclonal IgMκIIIb anti-I CA (protein KAU), proved capable of inhibiting human adult erythrocyte cryoagglutination by anti-I CAs but not that of fetal erythrocytes by anti-i CAs. Western blot analysis disclosed that such MoAbs recognized a sequential epitope located in the Fd fragment of all anti-I CAs employed in this study. With the purpose of checking whether Asp31 was involved in the epitope recognized by the MoAb, two peptides, D and G, were synthesized which mimicked the CDR1H structure of anti-I and anti-i, respectively; the MoAb only reacted with peptide D by ELISA. Subsequent experimental results indicate that the Gly/Asp mutation could be associated with the diverse specificity presented by these autoantibodies, a change determining a characteristic epitope/idiotope, recognized by LP91 in the CDR1H. PMID:8603526

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

Development of a blocking ELISA based on a monoclonal antibody against a predominant epitope in non-structural protein 3B2 of foot-and-mouth disease virus for differentiating infected from vaccinated animals.

PubMed

Fu, Yuanfang; Lu, Zengjun; Li, Pinghua; Cao, Yimei; Sun, Pu; Tian, Meina; Wang, Na; Bao, Huifang; Bai, Xingwen; Li, Dong; Chen, Yingli; Liu, Zaixin

2014-01-01

A monoclonal antibody (McAb) against non-structural protein (NSP) 3B of foot-mouth-disease virus (FMDV) (3B4B1) was generated and shown to recognize a conserved epitope spanning amino acids 24-32 of 3B (GPYAGPMER) by peptide screening ELISA. This epitope was further shown to be a unique and predominant B cell epitope in 3B2, as sera from animals infected with different serotypes of FMDV blocked the ability of McAb 3B4B1 to bind to NSP 2C3AB. Also, a polyclonal antibody against NSP 2C was produced in a rabbit vaccinated with 2C epitope regions expressed in E. coli. Using McAb 3B4B1 and the 2C polyclonal antibody, a solid-phase blocking ELISA (SPB-ELISA) was developed for the detection of antibodies against NSP 2C3AB to distinguish FMDV-infected from vaccinated animals (DIVA test). The parameters for this SPB-ELISA were established by screening panels of sera of different origins. Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal. This test showed a similar performance as the commercially available PrioCHECK NS ELISA. This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

PubMed

Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

2015-10-29

Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boonsathorn, Naphatsawan; Panthong, Sumolrat; Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development

Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutininmore » (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.« less

Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

PubMed

Gardyan, Adriane; Osen, Wolfram; Zörnig, Inka; Podola, Lilli; Agarwal, Maria; Aulmann, Sebastian; Ruggiero, Eliana; Schmidt, Manfred; Halama, Niels; Leuchs, Barbara; von Kalle, Christof; Beckhove, Philipp; Schneeweiss, Andreas; Jäger, Dirk; Eichmüller, Stefan B

2015-06-01

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. © 2014 UICC.

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

PubMed Central

van den Broeck, Hetty C; van Herpen, Teun WJM; Schuit, Cees; Salentijn, Elma MJ; Dekking, Liesbeth; Bosch, Dirk; Hamer, Rob J; Smulders, Marinus JM; Gilissen, Ludovicus JWJ; van der Meer, Ingrid M

2009-01-01

Background Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4+ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD). Results The effects of deleting individual gluten loci on both the level of T-cell stimulatory epitopes in the gluten proteome and the technological properties of the flour were analyzed using a set of deletion lines of Triticum aestivum cv. Chinese Spring. The reduction of T-cell stimulatory epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten proteins. The deletion lines were technologically tested with respect to dough mixing properties and dough rheology. The results show that removing the α-gliadin locus from the short arm of chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell stimulatory epitopes but also in a significant loss of technological properties. However, removing the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome (1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological properties. Conclusion The consequences of these data are discussed with regard to reducing the load of T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties. PMID:19351412

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

PubMed

James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

2008-05-15

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease

PubMed Central

de Jong, Hein C.; Salentijn, Elma M. J.; Dekking, Liesbeth; Bosch, Dirk; Hamer, Rob J.; Gilissen, Ludovicus J. W. J.; van der Meer, Ingrid M.; Smulders, Marinus J. M.

2010-01-01

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD toxic’ as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD. PMID:20664999

H1N1 viral proteome peptide microarray predicts individuals at risk for H1N1 infection and segregates infection versus Pandemrix® vaccination

PubMed Central

Ambati, Aditya; Valentini, Davide; Montomoli, Emanuele; Lapini, Guilia; Biuso, Fabrizio; Wenschuh, Holger; Magalhaes, Isabelle; Maeurer, Markus

2015-01-01

A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix® vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251–265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine. PMID:25639813

Thyroid peroxidase autoantibody fingerprints. II. A longitudinal study in postpartum thyroiditis.

PubMed

Jaume, J C; Parkes, A B; Lazarus, J H; Hall, R; Costante, G; McLachlan, S M; Rapoport, B

1995-03-01

It is not known whether epitopes recognized by autoantibodies in an individual remain constant or change over time, especially during perturbations of the humoral immune response. To address this question, we studied the epitopic profile ("fingerprint") of autoantibodies to thyroid peroxidase (TPO) in the sera of 19 women during the postpartum period. Fingerprints were determined in competition studies using 4 recombinant F(ab). At delivery and at 3 time intervals over the subsequent 9-12 months, the pool of F(ab) inhibited autoantibody binding to TPO by 80-100%, consistent with the definition by these F(ab) of a TPO immunodominant region (A1, A2, B1, and B2 domains). Despite a wide spectrum among individuals, the TPO epitopic fingerprints for all 19 women were relatively unchanged throughout the postpartum period. Fingerprint constancy occurred regardless of fluctuations in serum TPO autoantibody levels. When assessed numerically as a ratio of inhibition by the A domain F(ab) to inhibition by the B domain F(ab), the A/B domain ratios in individual women ranged from 0.2 (predominantly B domain) to more than 3.0 (predominantly A domain). However, for each individual woman, the A/B epitopic ratio was conserved throughout the study interval. Our TPO autoantibody epitopic fingerprint data have potential implications for understanding the humoral autoimmune response in man. First, the present study indicates a remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr. Second, and perhaps more important, despite marked variations in TPO epitopic profiles among different individuals, their constancy over time suggests that TPO autoantibody fingerprints may be inherited.

Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

PubMed Central

Calis, Jorg J. A.; Maybeno, Matt; Greenbaum, Jason A.; Weiskopf, Daniela; De Silva, Aruna D.; Sette, Alessandro; Keşmir, Can; Peters, Bjoern

2013-01-01

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses. PMID:24204222

Recognition of Naegleriae ameba surface protein epitopes by anti-human CD45 antibodies.

PubMed

Ravine, Terrence J; Polski, Jacek M; Jenkins, James

2010-04-01

Phagocytosis is a highly conserved mechanism exhibited by both free-living amebas and mammalian blood cells. Similarities demonstrated by either cell type during engulfment of the same bacterial species may imply analogous surface proteins involved in receptor-mediated endocytosis. The increased availability of anti-human leukocyte antibodies or clusters of differentiation (CD) markers used in conjunction with flow cytometric (FCM) and/or immunohistochemical (IHC) analysis provides investigators with a relatively easy method to screen different cell populations for comparable plasma membrane proteins. In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be considered when selecting potential mAbs for testing to increase the probability of a positive result.

Common T cell receptor clonotype in lacrimal glands and labial salivary glands from patients with Sjögren's syndrome.

PubMed Central

Matsumoto, I; Tsubota, K; Satake, Y; Kita, Y; Matsumura, R; Murata, H; Namekawa, T; Nishioka, K; Iwamoto, I; Saitoh, Y; Sumida, T

1996-01-01

Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS. PMID:8621782

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

NASA Technical Reports Server (NTRS)

He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

NASA Technical Reports Server (NTRS)

He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

PubMed Central

Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

2016-01-01

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shedlock, Devon J., E-mail: shedlock@mail.med.upenn.ed; Bailey, Michael A., E-mail: mike.bailey@taurigroup.co; Popernack, Paul M.

2010-06-05

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished bymore » reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.« less

T-cell memory responses elicited by yellow fever vaccine are targeted to overlapping epitopes containing multiple HLA-I and -II binding motifs.

PubMed

de Melo, Andréa Barbosa; Nascimento, Eduardo J M; Braga-Neto, Ulisses; Dhalia, Rafael; Silva, Ana Maria; Oelke, Mathias; Schneck, Jonathan P; Sidney, John; Sette, Alessandro; Montenegro, Silvia M L; Marques, Ernesto T A

2013-01-01

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4(+) and CD8(+) T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

PubMed Central

de Melo, Andréa Barbosa; Nascimento, Eduardo J. M.; Braga-Neto, Ulisses; Dhalia, Rafael; Silva, Ana Maria; Oelke, Mathias; Schneck, Jonathan P.; Sidney, John; Sette, Alessandro; Montenegro, Silvia M. L.; Marques, Ernesto T. A.

2013-01-01

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, “promiscuous” T-cell antigens might contribute to the high efficacy of the yellow fever vaccines. PMID:23383350

HIV-1 V3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features.

PubMed

Gazarian, Karlen G; Palacios-Rodríguez, Yadira; Gazarian, Tatiana G; Huerta, Leonor

2013-06-01

The crown region of the V3 loop in HIV-1 that contains the conserved amino acid sequence GPGR/G is known as the principal neutralizing determinant due to the extraordinary ability of antibodies to this region to neutralize the virus. To complement the existing peptide models of this epitope, we describe a family of 18 phage-displayed peptides, which include linear 12mer and constrained 7mer peptides that was selected by screening random libraries with serum from HIV-1 subtype B-infected patients. The 7mer constrained peptides presented two conserved amino acid sequences: PR-L in N-terminus and GPG in the C-terminus. On the basis of these peptides we propose a mimotope model of the V3 crown epitope in which the PR-L and GPG sequences represent the two known epitope binding sites. The GPG, has the same function as the V3 crown GPGR sequence but without the involvement of the "R" despite its being considered as the signature of the epitope in B-subtype viruses. The PR-L contains a proline not existing in the epitope that is postulated to induce kinks in the backbones of all peptides and create a spatial element mimicking the N-terminal conformationally variable binding site. Rabbit serum to these mimotopes recognized the V3 peptides and moderately decreased the fusion between HIV-1 Env- and CD4-expressing Jurkat cells. This study proposes the efficient generation by means of patient sera of V3 epitope mimics validated by interaction with the antibodies to contemporary viruses induced in patients. The serum antibody-selectable mimotopes are sources of novel information on the fine structure-function properties of HIV-1 principal neutralizing domain and candidate anti-HIV-1 immunogens. Copyright © 2012 Elsevier Ltd. All rights reserved.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

PubMed

Kessler, J H; Bres-Vloemans, S A; van Veelen, P A; de Ru, A; Huijbers, I J G; Camps, M; Mulder, A; Offringa, R; Drijfhout, J W; Leeksma, O C; Ossendorp, F; Melief, C J M

2006-10-01

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

PubMed

Smidt, Werner

2013-01-01

The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

PubMed

Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

2016-12-15

The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antibodies generated by immunization with the NS1 protein of West Nile virus confer partial protection against lethal Japanese encephalitis virus challenge.

PubMed

Sun, EnCheng; Zhao, Jing; TaoYang; Xu, QingYuan; Qin, YongLi; Wang, WenShi; Wei, Peng; Wu, DongLai

2013-09-27

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are two medically important flaviviruses that can cause severe hemorrhagic and encephalitic diseases in humans. Immune responses directed against the NS1 protein of flaviviruses can confer protection against lethal viral challenge. Previous studies have shown that the WNV NS1 protein harbors epitopes that elicit antibodies that cross react with JEV. Here we demonstrate that the WNV NS1 protein not only contains cross-reactive epitopes, but that the antibodies elicited by these cross-reactive epitopes provide partial protection against lethal JEV challenge in a mouse model. Mice immunized with WNV NS1 protein showed reduced morbidity and mortality following both intracerebral and intraperitoneal JEV challenge. WNV NS1 immunization attenuated the extent of lung pathology generated following JEV challenge, and delayed the appearance of other pathological findings including vascular cuffing. By screening and identifying the specific WNV NS1 protein-derived peptides recognized by serum antibodies elicited by immunization with WNV NS1 protein and by JEV challenge, we found after JEV challenge will induce several new epitopes, but which epitope primarily contribute to antibody-mediated cross protection need further evaluation. The knowledge and reagents generated in this study have potential applications in vaccine and subunit vaccine development for WNV and JEV. Copyright © 2013 Elsevier B.V. All rights reserved.

Evolutionarily Conserved Epitopes on Human Immunodeficiency Virus Type 1 (HIV-1) and Feline Immunodeficiency Virus Reverse Transcriptases Detected by HIV-1-Infected Subjects

PubMed Central

Sanou, Missa P.; Roff, Shannon R.; Mennella, Antony; Sleasman, John W.; Rathore, Mobeen H.; Levy, Jay A.

2013-01-01

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV+) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV+ subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8+ T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8+ T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV+ subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine. PMID:23824804

Ligand-induced Epitope Masking

PubMed Central

Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

2016-01-01

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

Type-specific and cross-reactive antibodies and T cell responses in norovirus VLP immunized mice are targeted both to conserved and variable domains of capsid VP1 protein.

PubMed

Malm, Maria; Tamminen, Kirsi; Vesikari, Timo; Blazevic, Vesna

2016-10-01

Norovirus (NoV)-specific antibodies, which block binding of the virus-like particles (VLPs) to the cell receptors are conformation dependent and directed towards the most exposed domain of the NoV capsid VP1 protein, the P2 domain. Limited data are available on the antibodies directed to other domains of the VP1, and even less on the NoV VP1-specific T cell epitopes. In here, BALB/c mice were immunized with six VLPs derived from NoV GII.4-1999, GII.4-2009 (New Orleans), GII.4-2012 (Sydney), GII.12, GI.1, and G1.3. Serum immunoglobulin G binding antibodies, histo-blood group antigen blocking antibodies and T cell responses using type-specific and heterologous NoV VLPs, P-dimers and 76 overlapping synthetic peptides, spanning the entire 539 amino acid sequence of GII.4 VP1, were determined. The results showed that at least half of the total antibody content is directed towards conserved S domain of the VP1. Only a small fraction (<1%) of the VP1 binding antibodies were blocking/neutralizing. With the use of matrix peptide pools and individual peptides, seven CD4 + and CD8 + T cell restricted epitopes were mapped, two located in S domain, four in P2 domain and one in P1 domain of NoV VP1. The epitopes were GII.4 strain-specific but also common GII.4 genotype-specific T cell epitopes were identified. More importantly, the results suggest a 9-amino acids long sequence ( 318 PAPLGTPDF 326 ) in P2 domain of VP1 as a universal NoV genogroup II-specific CD8 + T cell epitope. Distribution of the T cell epitopes alongside the capsid VP1 indicates the need of the complete protein for high immunogenicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

IgM-mediated autoimmune responses directed against anchorage epitopes are greater in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) than in major depression.

PubMed

Maes, Michael; Mihaylova, Ivana; Kubera, Marta; Leunis, Jean-Claude; Twisk, Frank N M; Geffard, Michel

2012-12-01

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and depression are considered to be neuro-immune disorders (Maes and Twisk, BMC Medicine 8:35, 2010). There is also evidence that depression and ME/CFS are accompanied by oxidative and nitrosative stress (O&NS) and by increased autoantibodies to a number of self-epitopes some of which have become immunogenic due to damage by O&NS. The aim of this study is to examine IgM-mediated autoimmune responses to different self-epitopes in ME/CFS versus depression. We examined serum IgM antibodies to three anchorage molecules (palmitic and myristic acid and S-farnesyl-L-cysteine); acetylcholine; and conjugated NO-modified adducts in 26 patients with major depression; 16 patients with ME/CFS, 15 with chronic fatigue; and 17 normal controls. Severity of fatigue and physio-somatic (F&S) symptoms was measured with the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale. Serum IgM antibodies to the three anchorage molecules and NO-phenylalanine were significantly higher in ME/CFS than in depression. The autoimmune responses to oxidatively, but not nitrosatively, modified self-epitopes were significantly higher in ME/CFS than in depression and were associated with F&S symptoms. The autoimmune activity directed against conjugated acetylcholine did not differ significantly between ME/CFS and depression, but was greater in the patients than controls. Partially overlapping pathways, i.e. increased IgM antibodies to a multitude of neo-epitopes, underpin both ME/CFS and depression, while greater autoimmune responses directed against anchorage molecules and oxidatively modified neo-epitopes discriminate patients with ME/CFS from those with depression. These autoimmune responses directed against neoantigenic determinants may play a role in the dysregulation of key cellular functions in both disorders, e.g. intracellular signal transduction, cellular differentiation and apoptosis, but their impact may be more important in ME/CFS than in depression.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence

PubMed Central

Wang, Yuge; Kapoor, Pratibha; Parks, Robert; Silva-Sanchez, Aaron; Alam, S. Munir; Verkoczy, Laurent; Liao, Hua-Xin; Zhuang, Yingxin; Burrows, Peter; Levinson, Michael; Elgavish, Ada; Cui, Xiangqin; Haynes, Barton F.; Schroeder, Harry

2015-01-01

Complementarity determining region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used ELISA or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity, but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germline sequence. This may help explain why antibodies in HIV infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization. PMID:26687685

Airway Memory CD4(+) T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses.

PubMed

Zhao, Jincun; Zhao, Jingxian; Mangalam, Ashutosh K; Channappanavar, Rudragouda; Fett, Craig; Meyerholz, David K; Agnihothram, Sudhakar; Baric, Ralph S; David, Chella S; Perlman, Stanley

2016-06-21

Two zoonotic coronaviruses (CoVs)-SARS-CoV and MERS-CoV-have crossed species to cause severe human respiratory disease. Here, we showed that induction of airway memory CD4(+) T cells specific for a conserved epitope shared by SARS-CoV and MERS-CoV is a potential strategy for developing pan-coronavirus vaccines. Airway memory CD4(+) T cells differed phenotypically and functionally from lung-derived cells and were crucial for protection against both CoVs in mice. Protection was dependent on interferon-γ and required early induction of robust innate and virus-specific CD8(+) T cell responses. The conserved epitope was also recognized in SARS-CoV- and MERS-CoV-infected human leukocyte antigen DR2 and DR3 transgenic mice, indicating potential relevance in human populations. Additionally, this epitope was cross-protective between human and bat CoVs, the progenitors for many human CoVs. Vaccine strategies that induce airway memory CD4(+) T cells targeting conserved epitopes might have broad applicability in the context of new CoVs and other respiratory virus outbreaks. Copyright © 2016 Elsevier Inc. All rights reserved.

Future of an “Asymptomatic” T-cell Epitope-Based Therapeutic Herpes Simplex Vaccine

PubMed Central

Dervillez, Xavier; Gottimukkala, Chetan; Kabbara, Khaled W.; Nguyen, Chelsea; Badakhshan, Tina; Kim, Sarah M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

2012-01-01

Summary Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as “asymptomatic” protective epitopes”) could boost local and systemic “natural” protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging “asymptomatic” T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease. PMID:22701511

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses.

PubMed

Molero-Abraham, Magdalena; Glutting, John-Paul; Flower, Darren R; Lafuente, Esther M; Reche, Pedro A

2015-01-01

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE PAGES

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; ...

2014-11-14

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses

PubMed Central

Molero-Abraham, Magdalena; Glutting, John-Paul; Flower, Darren R.; Lafuente, Esther M.; Reche, Pedro A.

2015-01-01

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes. PMID:26605344

Identification of three novel B-cell epitopes of VMH protein from Vibrio mimicus by screening a phage display peptide library.

PubMed

Xiao, Ning; Cao, Ji; Zhou, Hao; Ding, Shu-Quan; Kong, Ling-Yan; Li, Jin-Nian

2016-12-01

Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

A Subdominant CD8+ Cytotoxic T Lymphocyte (CTL) Epitope from the Plasmodium yoelii Circumsporozoite Protein Induces CTLs That Eliminate Infected Hepatocytes from Culture

PubMed Central

Franke, Eileen D.; Sette, Alessandro; Sacci, John; Southwood, Scott; Corradin, Giampietro; Hoffman, Stephen L.

2000-01-01

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57–70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58–67 sequence contains an H-2d binding motif, which binds purified Kd molecules in vitro with low affinity (3,267 nM) and encodes an H-2d-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57–70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59–67 specific, H-2d restricted, and CD8+ T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8+- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines. PMID:10816491

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

2016-06-01

HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

PubMed

Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

2011-07-18

The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

In vivo immunogenicity of Tax 11-19 epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine

PubMed Central

Sagar, Divya; Masih, Shet; Schell, Todd; Jacobson, Steven; Comber, Joseph D.; Philip, Ramila; Wigdahl, Brian; Jain, Pooja; Khan, Zafar K.

2014-01-01

Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund’s adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8 T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses. PMID:24739247

Structure of a High-Affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saphire, E.O.; Montero, M.; Menendez, A.

2007-07-13

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflectsmore » the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.« less

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

PubMed Central

Sedegah, Martha; Hollingdale, Michael R.; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Kim, Yohan; Peters, Bjoern; Sette, Alessandro; Huang, Jun; McGrath, Shannon; Abot, Esteban; Limbach, Keith; Shi, Meng; Soisson, Lorraine; Diggs, Carter; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E.; Villasante, Eileen; Richie, Thomas L.

2014-01-01

Background Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. Methodology/Principal Findings We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. Conclusions/Significance We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines. PMID:25211344

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

PubMed Central

Kitzmüller, Claudia; Zulehner, Nora; Roulias, Anargyros; Briza, Peter; Ferreira, Fatima; Faé, Ingrid; Fischer, Gottfried F.; Bohle, Barbara

2015-01-01

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing. PMID:25670010

Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

PubMed

Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

2013-01-01

We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

PubMed

Mendes, T M; Guimarães-Okamoto, P T C; Machado-de-Avila, R A; Oliveira, D; Melo, M M; Lobato, Z I; Kalapothakis, E; Chávez-Olórtegui, C

2015-06-01

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

PubMed

Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael

2017-06-08

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing

PubMed Central

Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E.; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael

2017-01-01

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates. PMID:28594355

Evidence of native α-synuclein conformers in the human brain.

PubMed

Gould, Neal; Mor, Danielle E; Lightfoot, Richard; Malkus, Kristen; Giasson, Benoit; Ischiropoulos, Harry

2014-03-14

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.

Immunochemical mapping of gonadotropins.

PubMed

Berger, P; Bidart, J M; Delves, P S; Dirnhofer, S; Hoermann, R; Isaacs, N; Jackson, A; Klonisch, T; Lapthorn, A; Lund, T; Mann, K; Roitt, I; Schwarz, S; Wick, G

1996-12-20

As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.

Targeted Gene Therapy for Breast Cancer

DTIC Science & Technology

1999-08-01

recognizing conformational and polysaccharide epitopes, cell ELISA using NIH3T3 and NIH/189 cells was employed to detect such clones. Cells (lxl04/well...77T 541-542, 1997. 26. Zhou, Y. and Lee, A. S. Mechanism for the suppression of the mammalian stress response by genistein, an anticancer

Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoetzel, Isidro; Cheevers, William P.

2005-09-01

The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains ofmore » CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.« less

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants*

PubMed Central

Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán

2015-01-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

PubMed

Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

2015-07-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Preparation and activity validation of PP7 bacteriophage-like particles displaying PAP114-128 peptide].

PubMed

Sun, Yanli; Sun, Yanhua

2016-10-01

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody.

PubMed

Ganesh, Vannakambadi K; Liang, Xiaowen; Geoghegan, Joan A; Cohen, Ana Luisa V; Venugopalan, Nagarajan; Foster, Timothy J; Hook, Magnus

2016-11-01

The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC 50 value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA. Copyright © 2016. Published by Elsevier B.V.

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping

PubMed Central

Stähle, I; Brizzio, C; Barile, M; Brandsch, R

1999-01-01

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (αFp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by αFp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to αFp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and αFP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-d-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize αFp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-d-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the αFp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by αFp-AB comprises, besides the flavin moiety, protein secondary structure elements. PMID:10193410

T cell epitope-specific defects in the immune response to cat allergen in patients with atopic dermatitis.

PubMed

Carneiro, Raquel; Reefer, Amanda; Wilson, Barbara; Hammer, Juergen; Platts-Mills, Thomas; Custis, Natalie; Woodfolk, Judith

2004-04-01

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes

PubMed Central

Nair, Smita K.; Tomaras, Georgia D.; Sales, Ana Paula; Boczkowski, David; Chan, Cliburn; Plonk, Kelly; Cai, Yongting; Dannull, Jens; Kepler, Thomas B.; Pruitt, Scott K.; Weinhold, Kent J.

2014-01-01

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19. PMID:24755960

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi

2009-09-11

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizingmore » ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.« less

Immunization of mice with baculovirus-derived recombinant SV40 large tumour antigen induces protective tumour immunity to a lethal challenge with SV40-transformed cells.

PubMed Central

Shearer, M H; Bright, R K; Lanford, R E; Kennedy, R C

1993-01-01

In this study, we examined the humoral immune responses and in vivo tumour immunity induced by baculovirus recombinant simian virus 40 (SV40) large tumour antigen (rSV40 T-ag). BALB/c mice immunized with rSV40 T-ag produced antibody responses that recognized SV40 large tumour antigen (T-ag) by ELISA. Analysis of these anti-SV40 T-ag responses indicated that the antibodies recognized epitopes associated with both the carboxy and amino terminus of SV40 T-ag. This pattern of SV40 T-ag epitope recognition was similar to that observed in anti-SV40 T-ag responses induced by inoculation with irradiated SV40-transformed cells. Mice immunized with either rSV40 T-ag or with the inactivated transformed cells were protected from a subsequent in vivo lethal tumour challenge with live SV40-transformed cells. These studies suggest that humoral immune responses induced by rSV40 T-ag are similar in epitope specificity to that induced by inactivated SV40-transformed cells. In addition, recombinant tumour-specific antigens from papovaviruses, such as SV40, can be used to induce tumour immunity which protects from a subsequent lethal tumour challenge. This study may provide insight into the use of recombinant tumour antigens as putative tumour vaccines and in the development of active immunotherapeutic strategies for treating virus-induced cancers. PMID:7679059

Detection of human antibodies binding with smooth and rough LPSs from Proteus mirabilis O3 strains S1959, R110, R45.

PubMed

Gleńska-Olender, J; Durlik, K; Konieczna, I; Kowalska, P; Gawęda, J; Kaca, W

2017-11-01

Bacteria of the genus Proteus of the family Enterobacteriaceae are facultative human pathogens responsible mainly for urinary tract and wound infections, bacteremia and the development of rheumatoid arthritis (RA). We have analyzed and compared by ELISA the titer of antibodies in plasmas of healthy individuals and in sera of rheumatoid arthritis patients recognizing a potential host cross-reactive epitope (lysine-galacturonic acid epitopes) present in Proteus lipopolysaccharide (LPS). In our experiments LPSs isolated from two mutants of smooth Proteus mirabilis 1959 (O3), i.e. strains R110 and R45, were used. R110 (Ra type mutant) is lacking the O-specific polysaccharide, but possesses a complete core oligosaccharide, while R45 (Re type) has a reduced core oligosaccharide and contains two 3-deoxy-D-manno-oct-2-ulosonic acid residues and one of 4-amino-4-deoxy-L-arabinopyranose residues. Titer of P. mirabilis S1959 LPS-specific-antibodies increased with the age of blood donors. RA and blood donors' sera contained antibodies against S and Ra and Re type of P. mirabilis O3 LPSs. Antibodies recognizing lysine-galacturonic acid epitopes of O3 LPS were detected by ELISA in some plasmas of healthy individuals and sera of rheumatoid arthritis patients. RA patients antibodies reacting with P. mirabilis S1959 S and R LPSs may indicate a potential role of anti-LPS antibodies in molecular mimicry in RA diseases.

Fine mapping of canine parvovirus B cell epitopes.

PubMed

López de Turiso, J A; Cortés, E; Ranz, A; García, J; Sanz, A; Vela, C; Casal, J I

1991-10-01

In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.

Broad-Spectrum Inhibition of HIV-1 by a Monoclonal Antibody Directed against a gp120-Induced Epitope of CD4

PubMed Central

Burastero, Samuele E.; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo

2011-01-01

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1. PMID:21818294

Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

PubMed

Burastero, Samuele E; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo

2011-01-01

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

High titers of autoantibodies to glutamate decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

PubMed Central

Hampe, Christiane S.; Maitland, Murray E.; Gilliam, Lisa K.; Thi Phan, Thanh-H.; Sweet, Ian R.; Radtke, Jared R.; Bota, Vasile; Ransom, Bruce R.; Hirsch, Irl B.

2014-01-01

Objective Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders and patients with type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesize that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. Methods We analyzed GAD65Ab titer, inhibition of GAD65 enzyme activity, and pattern of GAD65Ab epitopes in a cohort of type 1 diabetes patients (n=100) and correlated our findings with neurological symptoms and diseases. Results Fourty-three percent (43/100) of the patients had detectable GAD65Ab titers (median=400 U/ml, range: 142–250,000U/ml). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000–250,000 U/ml). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of five patients inhibited the enzyme activity significantly (by 34–55%). Three of these patients complained of muscle stiffness and pain, which was documented in two of these patients. Conclusions Based on our findings we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. PMID:23512385

Designed Ankyrin Repeat Proteins: A New Approach to Mimic Complex Antigens for Diagnostic Purposes?

PubMed Central

Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A.; Lacroix-Desmazes, Sebastien; Stadler, Beda M.; Horn, Michael P.

2013-01-01

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures. PMID:23626669

Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis.

PubMed

Valentini, Davide; Ferrara, Giovanni; Advani, Reza; Hallander, Hans O; Maeurer, Markus J

2015-07-01

Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

In vitro responses to avian influenza H5 by human CD4 T-cells*

PubMed Central

Cusick, Matthew F; Wang, Shuping; Eckels, David D

2009-01-01

To address the question of whether human T-cells are capable of recognizing novel isolates of influenza virus, in vitro responses to recombinant antigens and synthetic peptides derived from the sequences of H1, H3, and H5 were examined in a cohort of 64 individuals selected from a healthy blood donor population. Humans respond in vitro to H1 and H3 following exposure through natural infection and vaccination. Responses to H5 were well correlated with those to H1 or H3 and thus a significant repertoire of H5-responsive T-cells is present in many individuals; clear non-responders to H1, H3, and H5, however, do exist. Differences were observed in the cytokine responses to H1, H3, and H5; whereas both IL-2 and IFN-γ production characteristic of memory responses were observed for H1 and H3, H5-specific responses elicited primarily IL-2 and little or no IFN-γ consistent with a naïve T cell phenotype. Responses to all influenza HA were restricted by HLA-DR molecules. To address the structural basis for T-cell recognition of H1 and H5, overlapping synthetic peptides were used to identify epitopes and to determine whether recognition of H5 was limited to homologous sequences in H1, the most closely related HA phylogenetically. Although responses were generally correlated, no complete structural overlap was observed. These results suggest that helper T cell cross reactivity between different influenza strains may impart cross-protection to H5N1 strain of influenza. PMID:19841175

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

PubMed Central

Ascough, Stephanie; Ingram, Rebecca J.; Chu, Karen K.; Reynolds, Catherine J.; Musson, Julie A.; Doganay, Mehmet; Metan, Gökhan; Ozkul, Yusuf; Baillie, Les; Sriskandan, Shiranee; Moore, Stephen J.; Gallagher, Theresa B.; Dyson, Hugh; Williamson, E. Diane; Robinson, John H.; Maillere, Bernard; Boyton, Rosemary J.; Altmann, Daniel M.

2014-01-01

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified. PMID:24788397

Measles Virus Hemagglutinin epitopes immunogenic in natural infection and vaccination are targeted by broad or genotype-specific neutralizing monoclonal antibodies.

PubMed

Muñoz-Alía, Miguel Angel; Casasnovas, José M; Celma, María Luisa; Carabaña, Juan; Liton, Paloma B; Fernandez-Muñoz, Rafael

2017-05-15

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (k off ). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site.

PubMed

Cheng, Hao D; Grimm, Sebastian K; Gilman, Morgan Sa; Gwom, Luc Christian; Sok, Devin; Sundling, Christopher; Donofrio, Gina; Hedestam, Gunilla B Karlsson; Bonsignori, Mattia; Haynes, Barton F; Lahey, Timothy P; Maro, Isaac; von Reyn, C Fordham; Gorny, Miroslaw K; Zolla-Pazner, Susan; Walker, Bruce D; Alter, Galit; Burton, Dennis R; Robb, Merlin L; Krebs, Shelly J; Seaman, Michael S; Bailey-Kellogg, Chris; Ackerman, Margaret E

2018-03-08

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.

CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

PubMed

Musson, Julie A; Reynolds, Catherine J; Rinchai, Darawan; Nithichanon, Arnone; Khaenam, Prasong; Favry, Emmanuel; Spink, Natasha; Chu, Karen K Y; De Soyza, Anthony; Bancroft, Gregory J; Lertmemongkolchai, Ganjana; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M; Robinson, John H

2014-12-15

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients. Copyright © 2014 by The American Association of Immunologists, Inc.

Characterizing the interactions between a naturally primed immunoglobulin A and its conserved Bacteroides thetaiotaomicron species-specific epitope in gnotobiotic mice.

PubMed

Peterson, Daniel A; Planer, Joseph D; Guruge, Janaki L; Xue, Lai; Downey-Virgin, Whitt; Goodman, Andrew L; Seedorf, Henning; Gordon, Jeffrey I

2015-05-15

The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1-/-, or Myd88-/- mice. Comparison of gnotobiotic Rag1-/- mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterizing the Interactions between a Naturally Primed Immunoglobulin A and Its Conserved Bacteroides thetaiotaomicron Species-specific Epitope in Gnotobiotic Mice*

PubMed Central

Peterson, Daniel A.; Planer, Joseph D.; Guruge, Janaki L.; Xue, Lai; Downey-Virgin, Whitt; Goodman, Andrew L.; Seedorf, Henning; Gordon, Jeffrey I.

2015-01-01

The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1−/−, or Myd88−/− mice. Comparison of gnotobiotic Rag1−/− mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. PMID:25795776

The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

PubMed Central

Elvers, Karen T.; Geoghegan, Ivey; Shoemark, Debbie K.; Lampasona, Vito; Bingley, Polly J.; Williams, Alistair J.K.

2013-01-01

Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions. PMID:22966073

Identification of Optimal Epitopes for Plasmodium falciparum Rapid Diagnostic Tests That Target Histidine-Rich Proteins 2 and 3

PubMed Central

Lee, Nelson; Gatton, Michelle L.; Pelecanos, Anita; Bubb, Martin; Gonzalez, Iveth; Bell, David; Cheng, Qin

2012-01-01

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs. PMID:22259210

Cooperation between Strain-Specific and Broadly Neutralizing Responses Limited Viral Escape and Prolonged the Exposure of the Broadly Neutralizing Epitope

PubMed Central

Anthony, Colin; York, Talita; Bekker, Valerie; Matten, David; Selhorst, Philippe; Ferreria, Roux-Cil; Garrett, Nigel J.; Karim, Salim S. Abdool; Morris, Lynn; Wood, Natasha T.; Moore, Penny L.

2017-01-01

ABSTRACT V3-glycan-targeting broadly neutralizing antibodies (bNAbs) are a focus of HIV-1 vaccine development. Understanding the viral dynamics that stimulate the development of these antibodies can provide insights for immunogen design. We used a deep-sequencing approach, together with neutralization phenotyping, to investigate the rate and complexity of escape from V3-glycan-directed bNAbs compared to overlapping early strain-specific neutralizing antibody (ssNAb) responses to the V3/C3 region in donor CAP177. Escape from the ssNAb response occurred rapidly via an N334-to-N332 glycan switch, which took just 7.5 weeks to reach >50% frequency. In contrast, escape from the bNAbs was mediated via multiple pathways and took longer, with escape first occurring through an increase in V1 loop length, which took 46 weeks to reach 50% frequency, followed by an N332-to-N334 reversion, which took 66 weeks. Importantly, bNAb escape was incomplete, with contemporaneous neutralization observed up to 3 years postinfection. Both the ssNAb response and the bNAb response were modulated by the presence/absence of the N332 glycan, indicating an overlap between the two epitopes. Thus, selective pressure by ssNAbs to maintain the N332 glycan may have constrained the bNAb escape pathway. This slower and incomplete viral escape resulted in prolonged exposure of the bNAb epitope, which may in turn have aided the maturation of the bNAb lineage. IMPORTANCE The development of an HIV-1 vaccine is of paramount importance, and broadly neutralizing antibodies are likely to be a key component of a protective vaccine. The V3-glycan-targeting bNAb responses are among the most promising vaccine targets, as they are commonly elicited during infection. Understanding the interplay between viral evolution and the development of these antibodies provides insights that may guide immunogen design. Our work contrasted the dynamics of the early strain-specific antibodies and the later broadly neutralizing responses to a common Env target (V3C3), showing slower and more complex escape from bNAbs. Constrained bNAb escape, together with evidence of contemporaneous autologous virus neutralization, supports the proposal that prolonged exposure of the bNAb epitope enabled the maturation of the bNAb lineage. PMID:28679760

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

PubMed

Lee, William T; Jones, Derek D; Yates, Jennifer L; Winslow, Gary M; Davis, April D; Rudd, Robert J; Barron, Christopher T; Cowan, Cailyn

2016-12-01

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of Phage Display to Generate Conformation-Sensor Recombinant Antibodies

PubMed Central

Haque, Aftabul; Tonks, Nicholas K.

2013-01-01

We describe a phage display approach that we have previously used to generate conformation-sensor antibodies that recognize specifically and stabilize the oxidized, inactive conformation of protein tyrosine phosphatase 1B (PTP1B). We use a solution-based panning and screening strategy conducted in the presence of reduced active PTP1B, which enriches antibodies to epitopes unique to the oxidized form, while excluding antibodies that recognize epitopes common to oxidized and reduced forms of PTP1B. This strategy avoids conventional solid-phase immobilization, with its inherent potential for denaturation of the antigen. In addition, a functional screening strategy selects scFvs directly for their capacity for both specific binding and stabilization of the target enzyme in its inactive conformation. These conformation-specific scFvs illustrate that stabilization of oxidized PTP1B is an effective strategy to inhibit PTP1B function; it is possible that this approach may be applicable to the PTP family as a whole. Using this protocol, isolation and characterization of specific scFvs from immune responsive animals should take ~6 weeks. PMID:23154784

Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

PubMed

Alvarez-Navarro, Carlos; Cragnolini, Juan J; Dos Santos, Helena G; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A

2013-09-06

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

PubMed Central

Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

2013-01-01

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

Identification of an HLA-DPB1*0501 Restricted Melan-A/MART-1 Epitope Recognized by CD4+ T Lymphocytes: Prevalence for Immunotherapy in Asian Populations

PubMed Central

Meng, Zhaoting; Wang, Yadong; Zhang, Guanzhong; Ke, Yuehua; Yan, Yanfeng; Wu, Liangliang; Huang, Qianrong; Zeng, Gang; Wang, Yu; Ying, Han; Jiao, Shunchang

2015-01-01

Summary CD4+ T lymphocytes play a central role in orchestrating an efficient antitumor immune response. Much effort has been devoted in the identification of major histocompatibility complex class II eptiopes from different tumor-associated antigens. Melan-A/ MART-1 is expressed specifically in normal melanocytes and tumor cells of 75% to 100% of melanoma patients. Melan-A/MART-1 is considered as an attractive target for cancer immunotherapy. In the past, several human leukocyte antigen (HLA) class II restricted epitopes have been identified and characterized, including Melan-A/ MART-11-20 (HLA-DR11 restricted),Melan-A/MART-125-36 (HLA-DQ6 and HLA-DR3 restricted), Melan-A/MART-127-40 (HLA-DR1 restricted), Melan-A/MART-151-73 (HLA-DR4 restricted), Melan-A/ MART-191-110 (HLA-DR52 restricted), and Melan-A/MART-1100-111 (HLA-DR1 restricted). Owing to the infrequent expression of the above HLA class II alleles in Asian populations, immunotherapy using these defined Melan-A/MART-1 peptides could potentially only benefit a very small percentage of Asian melanoma patients. In this study, we established several CD4+ T-cell clones by in vitro stimulation of peripheral blood mononuclear cells from a healthy donor by a peptide pool of 28 to 30 amino acid long peptides spanning the entire Melan-A/MART-1 protein. These CD4+ T-cell clones recognized a peptide that is embedded within Melan-A/ MART-121-50, in a HLA-DPB1*0501 restricted manner. Finally, we demonstrated that this epitope is naturally processed and presented by dendritic cells. HLA-DPB1*0501 is frequently expressed in Asian population (44.9% to 73.1%). Therefore, this epitope could provide a new tool and could significantly increase the percentage of melanoma patients that can benefit from cancer immunotherapy. PMID:21760531

Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garrett, T.; Burgess, A; Gan, H

2009-01-01

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR{sub 287-302} complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR.more » However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR{sub C271A/C283A} mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR{sub C271A/C283A}. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.« less

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies.

PubMed

Sundaram, Roshni; Lynch, Marcus P; Rawale, Sharad V; Sun, Yiping; Kazanji, Mirdad; Kaumaya, Pravin T P

2004-06-04

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

Mutant MHC class II epitopes drive therapeutic immune responses to cancer.

PubMed

Kreiter, Sebastian; Vormehr, Mathias; van de Roemer, Niels; Diken, Mustafa; Löwer, Martin; Diekmann, Jan; Boegel, Sebastian; Schrörs, Barbara; Vascotto, Fulvia; Castle, John C; Tadmor, Arbel D; Schoenberger, Stephen P; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

2015-04-30

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

PubMed Central

Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Rosa Borges, Andrew; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

2012-01-01

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics. PMID:22970187

Analysis of Structures, Functions, and Epitopes of Cysteine Protease from Spirometra erinaceieuropaei Spargana

PubMed Central

Liu, Li Na; Cui, Jing; Zhang, Xi; Wei, Tong; Jiang, Peng; Wang, Zhong Quan

2013-01-01

Spirometra erinaceieuropaei cysteine protease (SeCP) in sparganum ES proteins recognized by early infection sera was identified by MALDI-TOF/TOF-MS. The aim of this study was to predict the structures and functions of SeCP protein by using the full length cDNA sequence of SeCP gene with online sites and software programs. The SeCP gene sequence was of 1 053 bp length with a 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidase C1A conserved domain. The predicted molecular weight and isoelectric point of SeCP were 37.87 kDa and 6.47, respectively. The SeCP has a signal peptide site and no transmembrane domain, located outside the membrane. The secondary structure of SeCP contained 8 α-helixes, 7 β-strands, and 20 coils. The SeCP had 15 potential antigenic epitopes and 19 HLA-I restricted epitopes. Based on the phylogenetic analysis of SeCP, S. erinaceieuropaei has the closest evolutionary status with S. mansonoides. SeCP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of antisparganum drugs. PMID:24392448

Thyroglobulin autoantibodies switch to immunoglobulin (Ig)G1 and IgG3 subclasses and preserve their restricted epitope pattern after 131I treatment for Graves' hyperthyroidism: the activity of autoimmune disease influences subclass distribution but not epitope pattern of autoantibodies

PubMed Central

Latrofa, F; Ricci, D; Montanelli, L; Piaggi, P; Mazzi, B; Bianchi, F; Brozzi, F; Santini, P; Fiore, E; Marinò, M; Tonacchera, M; Vitti, P

2014-01-01

The subclass distribution of thyroglobulin autoantibodies (TgAb) is debated, whereas their epitope pattern is restricted. Radioidine (131I) treatment for Graves' disease (GD) induces a rise in TgAb levels, but it is unknown whether it modifies subclass distribution and epitope pattern of TgAb as well. We collected sera from GD patients before 131I treatment and 3 and 6 months thereafter. We measured total TgAb, TgAb light chains and TgAb subclasses by enzyme-linked immunosorbent assay (ELISA) in 25 patients. We characterized the TgAb epitope pattern in 30 patients by inhibiting their binding to 125-ITg by a pool of four TgAb-Fab (recognizing Tg epitope regions A, B, C and D) and to Tg in ELISA by each TgAb-Fab. Total TgAb immunoglobulin (Ig)G rose significantly (P = 0·024). TgAb κ chains did not change (P = 0·052), whereas TgAb λ chains increased significantly (P = 0·001) and persistently. We observed a significant rise in IgG1 and IgG3 levels after 131I (P = 0·008 and P = 0·006, respectively), while IgG2 and IgG4 levels did not change. The rise of IgG1 was persistent, that of IgG3 transient. The levels of inhibition of TgAb binding to Tg by the TgAb-Fab pool were comparable. A slight, non-significant reduction of the inhibition by the immune-dominant TgAb-Fab A was observed 3 and 6 months after 131I. We conclude that 131I treatment for GD increases the levels of the complement-activating IgG1 and IgG3 subclasses and does not influence significantly the epitope pattern of TgAb. In autoimmune thyroid disease subclass distribution of autoantibodies is dynamic in spite of a stable epitope pattern. PMID:25134846

Structural basis for clonal diversity of the human T-cell response to a dominant influenza virus epitope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xinbo; Chen, Guobing; Weng, Nan-ping

Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8+ cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/β chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ~40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50)more » expressing the TRAV13-1/TRBV27 gene combination bound to GIL–HLA-A2 to 1.7 Å resolution. Comparison of the F50–GIL–HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL–HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide–MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50–GIL–HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide–MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.« less

Schistosoma mansoni Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

PubMed Central

Tremblay, Jacqueline M.; Oliveira, Sergio C.; Da’dara, Akram A.; Skelly, Patrick J.

2017-01-01

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development. PMID:28095417

CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

PubMed

Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

2013-02-01

CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

PubMed

Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J; Schneider, Pascal

2014-02-14

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia*

PubMed Central

Kowalczyk-Quintas, Christine; Willen, Laure; Dang, Anh Thu; Sarrasin, Heidi; Tardivel, Aubry; Hermes, Katharina; Schneider, Holm; Gaide, Olivier; Donzé, Olivier; Kirby, Neil; Headon, Denis J.; Schneider, Pascal

2014-01-01

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated. PMID:24391090

Induction of A. fumigatus-specific CD4-positive T cells in patients recovering from invasive aspergillosis

PubMed Central

Jolink, Hetty; Hagedoorn, Renate S.; Lagendijk, Ellen L.; Drijfhout, Jan W.; van Dissel, Jaap T.; Falkenburg, J.H. Frederik; Heemskerk, Mirjam H.M.

2014-01-01

After allogeneic stem cell transplantation patients are at risk of invasive aspergillosis, especially during the period of neutropenia. Recent data suggest that impaired T-cell immune reconstitution after transplantation plays an important role in this increased risk. In this study we investigated whether Aspergillus-specific T cells are involved in the recovery from invasive aspergillosis by analyzing the Aspergillus-specific T-cell response in patients with invasive aspergillosis. In nine patients whose Aspergillus infection improved, we identified Crf1- or Catalase1-specific T cells on the basis of CD154 expression and interferon-γ production following stimulation with overlapping peptides of the A. fumigatus proteins Crf1 and Catalase1. These Aspergillus-specific T cells were induced at the moment of regression of the aspergillus lesions. Crf1- and Catalase1-specific T cells, sorted on the basis of CD154 expression at the peak of the immune response, had a T helper-1 phenotype and recognized a variety of T-cell epitopes. In contrast, in two patients with progressive invasive aspergillosis, no Crf1- or Catalase1-specific T cells were identified. These data indicate that the presence of Aspergillus-specific T cells with a T helper-1 phenotype correlates with the clearance of aspergillus infection. PMID:24747947

The resistance mutation R155K in the NS3/4A protease of hepatitis C virus also leads the virus to escape from HLA-A*68-restricted CD8 T cells.

PubMed

Salloum, Shadi; Kluge, Silvia F; Kim, Arthur Y; Roggendorf, Michael; Timm, Joerg

2010-08-01

The NS3/4A serine protease of the hepatitis C virus (HCV) is one of the most attractive targets for specific antiviral agents. However, mutations conferring resistance may decrease the efficacy of these drugs. Although the level of resistance associated with specific mutations differs between different compounds, substitutions R155K and A156T reduce susceptibility to all protease inhibitors published so far. Interestingly, variants harboring the resistant mutation R155K were also detected as the predominant quasispecies in some treatment-naïve patients. Of note, key positions for resistance overlap with the HLA-A*68-restricted epitope HAVGIFRAAV(1175-1184). The aim of our study was to analyze the impact of protease inhibitor resistance mutations on the replication level and the antiviral CD8 T cell response against this HCV epitope. Our findings suggest that the R155K variant is associated with a relatively high replication level and with a substantial loss of cross-recognition by specific CD8 T cells targeting the epitope HAVGIFRAAV(1175-1184), providing a possible explanation for its existence in the absence of drug selection pressure. Copyright 2010 Elsevier B.V. All rights reserved.

The Structural Immunology of Antibody Protection against West Nile Virus

PubMed Central

Diamond, Michael S.; Pierson, Theodore C.; Fremont, Daved H.

2009-01-01

Summary Recent investigations of the interaction between the West Nile virus (WNV) envelope protein (E) and monoclonal antibodies (mAbs) have elucidated fundamental insights into the molecular mechanisms of neutralization. Structural studies have defined an epitope on the lateral ridge of domain III (DIII-lr) of the WNV E protein that is recognized by antibodies with the strongest neutralizing activity in vitro and in vivo. Antibodies that bind this epitope are highly potent because they efficiently block at a post-entry step of viral infection with relatively low virion occupancy requirements. In this review, we will discuss the structural, molecular, and immunologic basis for antibody-mediated protection against WNV, and its implications for novel therapeutic or vaccine strategies. PMID:18837784

P53 Immune Responses in Breast Cancer Patients: Assessment of CTL Recognizing the HLA-A2.1 Restricted, Wild-type Sequence p53 (264-272) Epitope; Frequencies of Tetramer+ T Cells Specific for the Wild-Type Sequence P53 (264-272) Peptide in the Circulation of Patients with Head and Neck Cancer; The Ability of Variant Peptides to Reverse the Nonresponsiveness of T Lymphocytes to the Wild-Type Sequence P53 (264-272) Epitope

DTIC Science & Technology

2002-10-01

This document contains three papers focusing on the analysis of anti-p53 cellular immune responses of breast, head, neck, and oral cancer patients...variants were generated by amino acid exchanges at positions 6 (6T) and 7 (7W) of the peptide. The 7W variant peptide has potential for immunotherapy of nonresponsive oral cancer patients.

Peptides and peptidomimetics as immunomodulators

PubMed Central

Gokhale, Ameya S; Satyanarayanajois, Seetharama

2014-01-01

Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically. PMID:25186605

New strategies for allergen T cell epitope identification: going beyond IgE

PubMed Central

Schulten, Véronique; Peters, Bjoern; Sette, Alessandro

2014-01-01

Background Type I allergy and allergic asthma are common diseases in the developed world associated with IgE antibodies and Th2 cell reactivity. To date, the only causative treatment for allergic disease is specific immunotherapy (SIT). Method Here, we review recent works from our laboratory focused on identifying human T cell epitopes associated with allergic disease and their potential use as biomarkers or therapeutic targets for SIT. In previous studies, we have mapped T cell epitopes associated with the major ten Timothy grass (Tg) allergens, defined on the basis of human IgE reactivity by ELISPOT. Results Interestingly, in about 33% of allergic donors no T cell epitopes from overlapping peptides spanning the entire sequences of these allergens were identified, despite vigorous T cell responses to the Tg extract. Using a bioinformatics-proteomic approach, we identified a set of 93 novel Tg proteins, many of which were found to elicit IL-5 production in T cells from allergic donors despite lacking IgE reactivity. Next, we assessed T cell responses to the novel Tg proteins in donors who had been treated with subcutaneous specific immunotherapy (SCIT). A subset of these proteins showed a strong reduction of IL-5 responses in donors who had received SCIT compared to allergic donors, which correlated with patient's self-reported improvement of allergic symptoms. Conclusion A bioinformatics-proteomic approach has successfully identified additional Tg-derived T cell targets independent of IgE reactivity. This method can be applied to other allergies potentially leading to the discovery of promising therapeutic targets for allergen-specific immunotherapy. PMID:25402674

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

PubMed

Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C

2015-01-01

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells

PubMed Central

Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E.; Wang, Rong-Fu; Wang, Helen Y.

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4+ T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4+ T cell-mediated immunotherapy in melanoma. PMID:25993655

Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

PubMed

Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

2015-01-01

Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5β1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

PubMed

Mould, A Paul; Askari, Janet A; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A; Humphries, Martin J

2016-09-30

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The in Vitro Antigenicity of Plasmodium vivax Rhoptry Neck Protein 2 (PvRON2) B- and T-Epitopes Selected by HLA-DRB1 Binding Profile

PubMed Central

López, Carolina; Yepes-Pérez, Yoelis; Díaz-Arévalo, Diana; Patarroyo, Manuel E.; Patarroyo, Manuel A.

2018-01-01

Malaria caused by Plasmodium vivax is a neglected disease which is responsible for the highest morbidity in both Americas and Asia. Despite continuous public health efforts to prevent malarial infection, an effective antimalarial vaccine is still urgently needed. P. vivax vaccine development involves analyzing naturally-infected patients' immune response to the specific proteins involved in red blood cell invasion. The P. vivax rhoptry neck protein 2 (PvRON2) is a highly conserved protein which is expressed in late schizont rhoptries; it interacts directly with AMA-1 and might be involved in moving-junction formation. Bioinformatics approaches were used here to select B- and T-cell epitopes. Eleven high-affinity binding peptides were selected using the NetMHCIIpan-3.0 in silico prediction tool; their in vitro binding to HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101 or HLA-DRB1*1302 was experimentally assessed. Four peptides (39152 (HLA-DRB1*04 and 11), 39047 (HLA-DRB1*07), 39154 (HLADRB1*13) and universal peptide 39153) evoked a naturally-acquired T-cell immune response in P. vivax-exposed individuals from two endemic areas in Colombia. All four peptides had an SI greater than 2 in proliferation assays; however, only peptides 39154 and 39153 had significant differences compared to the control group. Peptide 39047 was able to significantly stimulate TNF and IL-10 production while 39154 stimulated TNF production. Allele-specific peptides (but not the universal one) were able to stimulate IL-6 production; however, none induced IFN-γ production. The Bepipred 1.0 tool was used for selecting four B-cell epitopes in silico regarding humoral response. Peptide 39041 was the only one recognized by P. vivax-exposed individuals' sera and had significant differences concerning IgG subclasses; an IgG2 > IgG4 profile was observed for this peptide, agreeing with a protection-inducing role against P. falciparum and P. vivax as previously described for antigens such as RESA and MSP2. The bioinformatics results and in vitro evaluation reported here highlighted two T-cell epitopes (39047 and 39154) being recognized by memory cells and a B-cell epitope (39041) identified by P. vivax-exposed individuals' sera which could be used as potential candidates when designing a subunit-based vaccine. PMID:29868512

Array-Based Discovery of Aptamer Pairs

DTIC Science & Technology

2014-12-11

affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via...standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because...conventional selection methods preferentially yield aptamers that recognize a dominant “hot spot” epitope. Our 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions.

PubMed

Waxman, Elisa A; Duda, John E; Giasson, Benoit I

2008-07-01

Sensitive detection of alpha-synuclein (alpha-syn) pathology is important in the diagnosis of disorders like Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy and in providing better insights into the etiology of these diseases. Several monoclonal antibodies that selectively react with aggregated alpha-syn in pathological inclusions and reveal extensive and underappreciated alpha-syn pathology in the brains of diseased patients were previously reported by Duda et al. (Ann Neurol 52:205-210, 2002). We sought to characterize the specificity of some of these antibodies (Syn 505, Syn 506 and Syn 514); using C-terminal and N-terminal truncations of alpha-syn, all three antibodies were determined to require N-terminal epitopes that minimally comprise amino acids 2-4, but possibly extend to amino acid 12 of alpha-syn. The selectivity of these antibodies was further assessed using biochemical analysis of human brains and reactivity to altered recombinant alpha-syn proteins with duplication variants of amino acids 1-12. In addition, by expressing wild-type or a double mutant (E46K/A53T) of alpha-syn in cultured cells and by comparing their immunoreactivities to another antibody (SNL-4), which has a similar primary epitope, it was determined that Syn 505, Syn 506 and Syn 514 recognize conformational variants of alpha-syn that is enhanced by the presence of the double mutations. These studies indicate that antibodies Syn 505, Syn 506 and Syn 514 preferentially recognize N-terminal epitopes in complex conformations, consistent with the dramatic conformational change associated with the polymerization of alpha-synuclein into amyloid fibrils that form pathological inclusions.

Targeting malignant B cells with an immunotoxin against ROR1

PubMed Central

Baskar, Sivasubramanian; Wiestner, Adrian; Wilson, Wyndham H.; Pastan, Ira; Rader, Christoph

2012-01-01

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC50 = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers. PMID:22531447

Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus

PubMed Central

Green, Brett J.; Friend, Sherri; Beezhold, Donald H.

2012-01-01

Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients, causing infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy, which often does not enable species-specific identification. There is thus a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant form of the haemolysin terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and five strains of A. terreus, showed that three mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope, PSNEFE, while mAb 15B5 recognizes the epitope LYEGQFHS. Time-course studies showed that terrelysin expression was highest during early hyphal growth and dramatically decreased after mycelial expansion. Immunolocalization studies demonstrated that terrelysin was not only localized within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 °C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest that terrelysin may be a candidate biomarker for A. terreus infection. PMID:22160315

In silico and ex vivo approaches indicate immune pressure on capsid and non-capsid regions of coxsackie B viruses in the human system.

PubMed

Kundu, Rhiannon; Knight, Robin; Dunga, Meenakshi; Peakman, Mark

2018-01-01

Coxsackie B Virus (CBV) infection has been linked to the aetiology of type 1 diabetes (T1D) and vaccination has been proposed as prophylaxis for disease prevention. Serum neutralising antibodies and the presence of viral protein and RNA in tissues have been common tools to examine this potential disease relationship, whilst the role of anti-CBV cytotoxic T cell responses and their targets have not been studied. To address this knowledge gap, we augmented conventional HLA-binding predictive algorithm-based epitope discovery by cross-referencing epitopes with sites of positive natural selection within the CBV3 viral genome, identified using mixed effects models of evolution. Eight epitopes for the common MHC class I allele HLA-A*0201 occur at sites that appear to be positively selected. Furthermore, such epitopes span the viral genome, indicating that effective anti-viral responses may not be restricted to the capsid region. To assess the spectrum of IFNy responses in non-diabetic subjects and recently diagnosed type 1 diabetes (T1D) patients, we stimulated PBMC ex vivo with pools of synthetic peptides based on component-restricted sequences identified in silico. We found responders were more likely to recognize multiple rather than a single CBV peptide pool, indicating that the natural course of infection results in multiple targets for effector memory responses, rather than immunodominant epitopes or viral components. The finding that anti-CBV CD8 T cell immunity is broadly targeted has implications for vaccination strategies and studies on the pathogenesis of CBV-linked diseases.

Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages

PubMed Central

Hua, Chun-Zhen; Hu, Wei-Lin; Li, Jian-Ping; Hong, Li-Quan

2015-01-01

Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae. PMID:26677200

Immunization with Hypoallergens of shrimp allergen tropomyosin inhibits shrimp tropomyosin specific IgE reactivity.

PubMed

Wai, Christine Y Y; Leung, Nicki Y H; Ho, Marco H K; Gershwin, Laurel J; Shu, Shang An; Leung, Patrick S C; Chu, Ka Hou

2014-01-01

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.

High-resolution crystal structure and IgE recognition of the major grass pollen allergen Phl p 3.

PubMed

Devanaboyina, S C; Cornelius, C; Lupinek, C; Fauland, K; Dall'Antonia, F; Nandy, A; Hagen, S; Flicker, S; Valenta, R; Keller, W

2014-12-01

Group 2 and 3 grass pollen allergens are major allergens with high allergenic activity and exhibit structural similarity with the C-terminal portion of major group 1 allergens. In this study, we aimed to determine the crystal structure of timothy grass pollen allergen, Phl p 3, and to study its IgE recognition and cross-reactivity with group 2 and group 1 allergens. The three-dimensional structure of Phl p 3 was solved by X-ray crystallography and compared with the structures of group 1 and 2 grass pollen allergens. Cross-reactivity was studied using a human monoclonal antibody which inhibits allergic patients' IgE binding and by IgE inhibition experiments with patients' sera. Conformational Phl p 3 IgE epitopes were predicted with the algorithm SPADE, and Phl p 3 variants containing single point mutations in the predicted IgE binding sites were produced to analyze allergic patients' IgE binding. Phl p 3 is a globular β-sandwich protein showing structural similarity to Phl p 2 and the Phl p 1-C-terminal domain. Phl p 3 showed IgE cross-reactivity with group 2 allergens but not with group 1 allergens. SPADE identified two conformational IgE epitope-containing areas, of which one overlaps with the epitope defined by the monoclonal antibody. The mutation of arginine 68 to alanine completely abolished binding of the blocking antibody. This mutation and a mutation of D13 in the predicted second IgE epitope area also reduced allergic patients' IgE binding. Group 3 and group 2 grass pollen allergens are cross-reactive allergens containing conformational IgE epitopes. They lack relevant IgE cross-reactivity with group 1 allergens and therefore need to be included in diagnostic tests and allergen-specific treatments in addition to group 1 allergens. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sequence conservation predicts T cell reactivity against ragweed allergens.

PubMed

Pham, J; Oseroff, C; Hinz, D; Sidney, J; Paul, S; Greenbaum, J; Vita, R; Phillips, E; Mallal, S; Peters, B; Sette, A

2016-09-01

Ragweed is a major cause of seasonal allergy, affecting millions of people worldwide. Several allergens have been defined based on IgE reactivity, but their relative immunogenicity in terms of T cell responses has not been studied. We comprehensively characterized T cell responses from atopic, ragweed-allergic subjects to Amb a 1, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10, Amb a 11, and Amb p 5 and examined their correlation with serological reactivity and sequence conservation in other allergens. Peripheral blood mononuclear cells (PBMCs) from donors positive for IgE towards ragweed extracts after in vitro expansion for secretion of IL-5 (a representative Th2 cytokine) and IFN-γ (Th1) in response to a panel of overlapping peptides spanning the above-listed allergens were assessed. Three previously identified dominant T cell epitopes (Amb a 1 176-191, 200-215, and 344-359) were confirmed, and three novel dominant epitopes (Amb a 1 280-295, 304-319, and 320-335) were identified. Amb a 1, the dominant IgE allergen, was also the dominant T cell allergen, but dominance patterns for T cell and IgE responses for the other ragweed allergens did not correlate. Dominance for T cell responses correlated with conservation of ragweed epitopes with sequences of other well-known allergens. These results provide the first assessment of the hierarchy of T cell reactivity in ragweed allergens, which is distinct from that observed for IgE reactivity and influenced by T cell epitope sequence conservation. The results suggest that ragweed allergens associated with lesser IgE reactivity and significant T cell reactivity may be targeted for T cell immunotherapy, and further support the development of immunotherapies against epitopes conserved across species to generate broad reactivity against many common allergens. © 2016 John Wiley & Sons Ltd.

Fine specificities of natural regulatory T cells after IVIG therapy in patients with Kawasaki disease

PubMed Central

Burns, Jane C.; Touma, Ranim; Song, Yali; Padilla, Robert L.; Tremoulet, Adriana H.; Sidney, John; Sette, Alessandro; Franco, Alessandra

2016-01-01

The activation of natural regulatory T cells (nTreg) recognizing the heavy constant region (Fc) of IgG is an important mechanism of action of intravenous immunoglobulin (IVIG) therapy in Kawasaki disease (KD). Lack of circulating Fc-specific nTreg in the sub-acute phase of KD is correlated with the development of coronary artery abnormalities (CAA). Here, we characterize the fine specificity of nTreg in sub-acute (2- to 8-week post-IVIG) and convalescent (1- to 10-year post-IVIG) KD subjects by testing the immunogenicity of 64 peptides, 15 amino acids in length with a 10 amino acid-overlap spanning the entire Fc protein. About 12 Fc peptides (6 pools of 2 consecutive peptides) were recognized by nTreg in the cohorts studied, including two patients with CAA. To test whether IVIG expands the same nTreg populations that maintain vascular homeostasis in healthy subjects, we compared these results with results obtained in healthy adult controls. Similar nTreg fine specificities were observed in KD patients after IVIG and in healthy donors. These results suggest that T cell fitness rather than T cell clonal deletion or anergy is responsible for the lack of Fc-specific nTreg in KD patients who develop CAA. Furthermore, we found that adolescents and adults who had KD during childhood without developing CAA did not respond to the Fc protein in vitro, suggesting that the nTreg response induced by IVIG in KD patients is short-lived. Our results support the concept that peptide epitopes may be a viable therapeutic approach to expand Fc-specific nTreg and more effectively prevent CAA in KD patients. PMID:25822882

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species.

PubMed

Yu, Meng; Stevens, Vicky; Berry, Jody D; Crameri, Gary; McEachern, Jennifer; Tu, Changchun; Shi, Zhengli; Liang, Guodong; Weingartl, Hana; Cardosa, Jane; Eaton, Bryan T; Wang, Lin-Fa

2008-02-29

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.

A study of the epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison's disease

PubMed Central

Volpato, M; Prentice, L; Chen, S; Betterle, C; Rees Smith, B; Furmaniak, J

1998-01-01

Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH. PMID:9486414

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brodzik, R.; Bandurska, K.; Deka, D.

2005-12-16

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles weremore » efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.« less

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana.

PubMed

Pereira, L A R; Schoor, S; Goubet, F; Dupree, P; Moffatt, B A

2006-11-01

Pectin methyl-esterification is catalysed by S-adenosyl-L: -methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.

Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes.

PubMed

He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

2017-07-15

Bovine β-lactoglobulin (BLG) is the main allergen in cows' milk, and the most commonly used method for detecting BLG is enzyme-linked immunosorbent assay (ELISA). However, antibodies used in commercial ELISA kits do not recognize specifically BLG IgE epitopes. Here, an antibody specific to IgE linear epitopes for BLG was used to develop a sandwich ELISA using a rabbit anti-BLG polyclonal antibody. The linear range for BLG detection was 31.25-8000ng/mL and limit of detection was 1.96ng/mL. BLG content in dairy samples was determined, and there was a good agreement between this immunoassay and reversed-phase high-performance liquid chromatography with high recovery. Additionally, BLG content in food samples had an average recovery of 104.25%. Allergenic residues were also detected in hydrolyzed infant formulas. The method developed could be a practical approach to determine BLG and its allergenic residues in food with a high degree of sensitivity, reliability and recovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Non-native Soluble Oligomers of Cu/Zn Superoxide Dismutase (SOD1) Contain a Conformational Epitope Linked to Cytotoxicity in Amyotrophic Lateral Sclerosis (ALS)

PubMed Central

2015-01-01

Soluble misfolded Cu/Zn superoxide dismutase (SOD1) is implicated in motor neuron death in amyotrophic lateral sclerosis (ALS); however, the relative toxicities of the various non-native species formed by SOD1 as it misfolds and aggregates are unknown. Here, we demonstrate that early stages of SOD1 aggregation involve the formation of soluble oligomers that contain an epitope specific to disease-relevant misfolded SOD1; this epitope, recognized by the C4F6 antibody, has been proposed as a marker of toxic species. Formation of potentially toxic oligomers is likely to be exacerbated by an oxidizing cellular environment, as evidenced by increased oligomerization propensity and C4F6 reactivity when oxidative modification by glutathione is present at Cys-111. These findings suggest that soluble non-native SOD1 oligomers, rather than native-like dimers or monomers, share structural similarity to pathogenic misfolded species found in ALS patients and therefore represent potential cytotoxic agents and therapeutic targets in ALS. PMID:24660965

Antibody Recognition of a Highly Conserved Influenza Virus Epitope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André

2009-05-21

Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes amore » highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.« less

Predicting 3D pose in partially overlapped X-ray images of knee prostheses using model-based Roentgen stereophotogrammetric analysis (RSA).

PubMed

Hsu, Chi-Pin; Lin, Shang-Chih; Shih, Kao-Shang; Huang, Chang-Hung; Lee, Chian-Her

2014-12-01

After total knee replacement, the model-based Roentgen stereophotogrammetric analysis (RSA) technique has been used to monitor the status of prosthetic wear, misalignment, and even failure. However, the overlap of the prosthetic outlines inevitably increases errors in the estimation of prosthetic poses due to the limited amount of available outlines. In the literature, quite a few studies have investigated the problems induced by the overlapped outlines, and manual adjustment is still the mainstream. This study proposes two methods to automate the image processing of overlapped outlines prior to the pose registration of prosthetic models. The outline-separated method defines the intersected points and segments the overlapped outlines. The feature-recognized method uses the point and line features of the remaining outlines to initiate registration. Overlap percentage is defined as the ratio of overlapped to non-overlapped outlines. The simulated images with five overlapping percentages are used to evaluate the robustness and accuracy of the proposed methods. Compared with non-overlapped images, overlapped images reduce the number of outlines available for model-based RSA calculation. The maximum and root mean square errors for a prosthetic outline are 0.35 and 0.04 mm, respectively. The mean translation and rotation errors are 0.11 mm and 0.18°, respectively. The errors of the model-based RSA results are increased when the overlap percentage is beyond about 9%. In conclusion, both outline-separated and feature-recognized methods can be seamlessly integrated to automate the calculation of rough registration. This can significantly increase the clinical practicability of the model-based RSA technique.

Autoimmune-autoinflammatory rheumatoid arthritis overlaps: a rare but potentially important subgroup of diseases

PubMed Central

Savic, Sinisa; Mistry, Anoop; Wilson, Anthony G; Barcenas-Morales, Gabriela; Doffinger, Rainer; Emery, Paul; McGonagle, Dennis

2017-01-01

At the population level, rheumatoid arthritis (RA) is generally viewed as autoimmune in nature with a small subgroup of cases having a palindromic form or systemic autoinflammatory disorder (SAID) phenotype. Herein, we describe resistant cases of classical autoantibody associated RA that had clinical, genetic and therapeutic responses indicative of coexistent autoinflammatory disease. Five patients with clinically overlapping features between RA and SAID including polysynovitis and autoantibody/shared epitope positivity, and who had abrupt severe self-limiting attacks including fevers and serositis, are described. Mutations or single nucleotide polymorphisms in recognised autoinflammatory pathways were evident. Generally, these cases responded poorly to conventional Disease-modifying anti-rheumatic drugs (DMARD) treatment with some excellent responses to colchicine or interleukin 1 pathway blockade. A subgroup of RA cases have a mixed autoimmune-autoinflammatory phenotype and genotype with therapeutic implications. PMID:29177082

Autoimmune-autoinflammatory rheumatoid arthritis overlaps: a rare but potentially important subgroup of diseases.

PubMed

Savic, Sinisa; Mistry, Anoop; Wilson, Anthony G; Barcenas-Morales, Gabriela; Doffinger, Rainer; Emery, Paul; McGonagle, Dennis

2017-01-01

At the population level, rheumatoid arthritis (RA) is generally viewed as autoimmune in nature with a small subgroup of cases having a palindromic form or systemic autoinflammatory disorder (SAID) phenotype. Herein, we describe resistant cases of classical autoantibody associated RA that had clinical, genetic and therapeutic responses indicative of coexistent autoinflammatory disease. Five patients with clinically overlapping features between RA and SAID including polysynovitis and autoantibody/shared epitope positivity, and who had abrupt severe self-limiting attacks including fevers and serositis, are described. Mutations or single nucleotide polymorphisms in recognised autoinflammatory pathways were evident. Generally, these cases responded poorly to conventional Disease-modifying anti-rheumatic drugs (DMARD) treatment with some excellent responses to colchicine or interleukin 1 pathway blockade. A subgroup of RA cases have a mixed autoimmune-autoinflammatory phenotype and genotype with therapeutic implications.