Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections.

PubMed

Capelli, Riccardo; Matterazzo, Elena; Amabili, Marco; Peri, Claudio; Gori, Alessandro; Gagni, Paola; Chiari, Marcella; Lertmemongkolchai, Ganjana; Cretich, Marina; Bolognesi, Martino; Colombo, Giorgio; Gourlay, Louise J

2017-10-13

Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.

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.

Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

PubMed Central

Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

2014-01-01

ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an HIV protein partially determines which epitopes are dominant, most likely by controlling the breakdown of HIV into peptides. Moreover, some types of signals from CD4+ T cells are affected by the HIV protein 3D structure; and thus the protectiveness of a particular peptide vaccine could be related to its location in the 3D structure. PMID:24920818

[Conformational Fingerprinting Using Monoclonal Antibodies
(on the Example of Angiotensin I-Converting Enzyme-ACE)].

PubMed

Danilov, S M

2017-01-01

During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept - conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

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

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.

Characterization of neutralizing epitopes of varicella-zoster virus glycoprotein H.

PubMed

Akahori, Yasushi; Suzuki, Kazuhiro; Daikoku, Tohru; Iwai, Masae; Yoshida, Yoshihiro; Asano, Yoshizo; Kurosawa, Yoshikazu; Shiraki, Kimiyasu

2009-02-01

Varicella-zoster virus (VZV) glycoprotein H (gH) is the major neutralization target of VZV, and its neutralizing epitope is conformational. Ten neutralizing human monoclonal antibodies to gH were used to map the epitopes by immunohistochemical analysis and were categorized into seven epitope groups. The combinational neutralization efficacy of two epitope groups was not synergistic. Each epitope was partially or completely resistant to concanavalin A blocking of the glycomoiety of gH, and their antibodies inhibited the cell-to-cell spread of infection. The neutralization epitope comprised at least seven independent protein portions of gH that served as the target to inhibit cell-to-cell spread.

Conformational analysis of the ΜΒΡ83-99 (Phe91) and ΜΒΡ83-99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using Molecular Dynamics

NASA Astrophysics Data System (ADS)

Potamitis, C.; Matsoukas, M.-T.; Tselios, T.; Mavromoustakos, T.; Golič Grdadolnik, S.

2011-09-01

The two new synthetic analogues of the MBP83-99 epitope substituted at Lys91 (primary TCR contact) with Phe [MBP83-99 (Phe91)] or Tyr [MBP83-99 (Tyr91)], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP83-99 epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP83-99 immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.

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.

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

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies*

PubMed Central

Dutta, Kaushik; Varshney, Avanish K.; Franklin, Matthew C.; Goger, Michael; Wang, Xiaobo; Fries, Bettina C.

2015-01-01

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations. PMID:25572397

Mechanisms mediating enhanced neutralization efficacy of Staphylococcal enterotoxin B by combinations of monoclonal antibodies

DOE PAGES

Dutta, Kaushik; Varshney, Avanish K.; Franklin, Matthew C.; ...

2015-01-08

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used tomore » validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Lastly structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.« less

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

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

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.

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.

Residue-Specific Side-Chain Polymorphisms via Particle Belief Propagation.

PubMed

Ghoraie, Laleh Soltan; Burkowski, Forbes; Li, Shuai Cheng; Zhu, Mu

2014-01-01

Protein side chains populate diverse conformational ensembles in crystals. Despite much evidence that there is widespread conformational polymorphism in protein side chains, most of the X-ray crystallography data are modeled by single conformations in the Protein Data Bank. The ability to extract or to predict these conformational polymorphisms is of crucial importance, as it facilitates deeper understanding of protein dynamics and functionality. In this paper, we describe a computational strategy capable of predicting side-chain polymorphisms. Our approach extends a particular class of algorithms for side-chain prediction by modeling the side-chain dihedral angles more appropriately as continuous rather than discrete variables. Employing a new inferential technique known as particle belief propagation, we predict residue-specific distributions that encode information about side-chain polymorphisms. Our predicted polymorphisms are in relatively close agreement with results from a state-of-the-art approach based on X-ray crystallography data, which characterizes the conformational polymorphisms of side chains using electron density information, and has successfully discovered previously unmodeled conformations.

Induced secondary structure and polymorphism in an intrinsically disordered structural linker of the CNS: solid-state NMR and FTIR spectroscopy of myelin basic protein bound to actin.

PubMed

Ahmed, Mumdooh A M; Bamm, Vladimir V; Shi, Lichi; Steiner-Mosonyi, Marta; Dawson, John F; Brown, Leonid; Harauz, George; Ladizhansky, Vladimir

2009-01-01

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully (13)C,(15)N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in beta-sheet content in actin, and increases in both alpha-helix and beta-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both alpha-helical and beta-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.

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.

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

PubMed

Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

2015-05-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

PubMed Central

Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

2015-01-01

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

Conformational polymorphs of a novel TCNQ derivative carrying an acetylene group

NASA Astrophysics Data System (ADS)

Iida, Yuki; Kataoka, Makoto; Okuno, Tsunehisa

2018-01-01

TCNQ is one of the most important organic acceptors and lots of its derivatives have been prepared. However the reports on their crystal polymorphs are limited to their complexes, and simple polymorphs of TCNQ derivatives are uncommon. We succeeded in preparation of a novel TCNQ derivative, 2,2'-(2-(prop-2-yn-1-yloxy)cyclohexa-2,5-diene-1,4-diylidene)dimalononitrile, having a propynyloxy group on a substituent. This compound was found to have two crystal polymorphs depending on a solvent for recrystallization. In polymorph I, dimeric hydrogen bonds are formed between acetylenic hydrogens and cyano nitrogens with the molecule in an inversion symmetry. While, in polymorph II, the molecules make intermolecular hydrogen bonds between acetylenic hydrogens and cyano nitrogens with the molecule in 21 symmetry, forming a hydrogen bonded molecular helix along the b axis. Besides patterns of the intermolecular hydrogen bonds, difference was recognized in conformation of propynyloxy group. The molecule has an anti conformation in polymorph I and a gauche conformation in polymorph II. DFT calculation indicates that the anti conformer is less stable than the gauche one. But a solvation model suggests the anti conformer is estimated to be more stable in a toluene solution.

Application of phage peptide display technology for the study of food allergen epitopes.

PubMed

Chen, Xueni; Dreskin, Stephen C

2017-06-01

Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pathological conformations involving the amino terminus of tau occur early in Alzheimer’s disease and are differentially detected by monoclonal antibodies

PubMed Central

Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M.

2016-01-01

Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer’s disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies’ relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7–12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer’s disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological changes in tau indicating that detection of conformational alterations involving PAD exposure is not achieved by all N-terminal tau antibodies and that a relatively discrete region of the N-terminus (i.e., amino acids 7–12, the TNT1 and TNT2 epitope) is central to the differences between normal and pathological tau. The appearance of PAD in early tau pathology and its disappearance in late-stage tangles suggest that toxic forms of tau are associated with the earliest forms of tau deposits. Collectively, these findings demonstrate that the TNT antibodies are useful markers for early conformational display of PAD and provide information regarding conformational changes that have potential implications in the toxic mechanisms of tau pathology. PMID:27260838

Proof of principle for epitope-focused vaccine design

PubMed Central

Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Christopher; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

2014-01-01

Summary Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Multiple major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus (RSV), that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for research and development of a human RSV vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets including antigenically highly variable pathogens such as HIV and influenza. PMID:24499818

Proof of principle for epitope-focused vaccine design

NASA Astrophysics Data System (ADS)

Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

2014-03-01

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Native Mass Spectrometry, Ion mobility, and Collision-Induced Unfolding Categorize Malaria Antigen/Antibody Binding

NASA Astrophysics Data System (ADS)

Huang, Yining; Salinas, Nichole D.; Chen, Edwin; Tolia, Niraj H.; Gross, Michael L.

2017-09-01

Plasmodium vivax Duffy Binding Protein (PvDBP) is a promising vaccine candidate for P. vivax malaria. Recently, we reported the epitopes on PvDBP region II (PvDBP-II) for three inhibitory monoclonal antibodies (2D10, 2H2, and 2C6). In this communication, we describe the combination of native mass spectrometry and ion mobility (IM) with collision induced unfolding (CIU) to study the conformation and stabilities of three malarial antigen-antibody complexes. These complexes, when collisionally activated, undergo conformational changes that depend on the location of the epitope. CIU patterns for PvDBP-II in complex with antibody 2D10 and 2H2 are highly similar, indicating comparable binding topology and stability. A different CIU fingerprint is observed for PvDBP-II/2C6, indicating that 2C6 binds to PvDBP-II on an epitope different from 2D10 and 2H2. This work supports the use of CIU as a means of classifying antigen-antibody complexes by their epitope maps in a high throughput screening workflow. [Figure not available: see fulltext.

Conformational flexibility and packing plausibility of repaglinide polymorphs

NASA Astrophysics Data System (ADS)

Rani, Dimpy; Goyal, Parnika; Chadha, Renu

2018-04-01

The present manuscript highlights the structural insight into the repaglinide polymorphs. The experimental screening for the possible crystal forms were carried out using various solvents, which generated three forms. The crystal structure of Form II and III was determined using PXRD pattern whereas structural analysis of Form I has already been reported. Form I, II and II was found to exist in P212121, PNA21 and P21/c space groups respectively. Conformational analysis was performed to account the conformational flexibility of RPG. The obtained conformers were further utilized to obtain the information about the crystal packing pattern of RPG polymorphs by polymorph prediction module. The lattice energy landscape, depicting the relationship between lattice energy and density of the polymorphs has been obtained for various possible polymorphs. The experimentally isolated polymorphs were successfully fitted into lattice energy landscape.

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.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen.

PubMed

Untersmayr, Eva; Szalai, Krisztina; Riemer, Angelika B; Hemmer, Wolfgang; Swoboda, Ines; Hantusch, Brigitte; Schöll, Isabella; Spitzauer, Susanne; Scheiner, Otto; Jarisch, Reinhart; Boltz-Nitulescu, George; Jensen-Jarolim, Erika

2006-03-01

Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.

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.

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

Ab-initio conformational epitope structure prediction using genetic algorithm and SVM for vaccine design.

PubMed

Moghram, Basem Ameen; Nabil, Emad; Badr, Amr

2018-01-01

T-cell epitope structure identification is a significant challenging immunoinformatic problem within epitope-based vaccine design. Epitopes or antigenic peptides are a set of amino acids that bind with the Major Histocompatibility Complex (MHC) molecules. The aim of this process is presented by Antigen Presenting Cells to be inspected by T-cells. MHC-molecule-binding epitopes are responsible for triggering the immune response to antigens. The epitope's three-dimensional (3D) molecular structure (i.e., tertiary structure) reflects its proper function. Therefore, the identification of MHC class-II epitopes structure is a significant step towards epitope-based vaccine design and understanding of the immune system. In this paper, we propose a new technique using a Genetic Algorithm for Predicting the Epitope Structure (GAPES), to predict the structure of MHC class-II epitopes based on their sequence. The proposed Elitist-based genetic algorithm for predicting the epitope's tertiary structure is based on Ab-Initio Empirical Conformational Energy Program for Peptides (ECEPP) Force Field Model. The developed secondary structure prediction technique relies on Ramachandran Plot. We used two alignment algorithms: the ROSS alignment and TM-Score alignment. We applied four different alignment approaches to calculate the similarity scores of the dataset under test. We utilized the support vector machine (SVM) classifier as an evaluation of the prediction performance. The prediction accuracy and the Area Under Receiver Operating Characteristic (ROC) Curve (AUC) were calculated as measures of performance. The calculations are performed on twelve similarity-reduced datasets of the Immune Epitope Data Base (IEDB) and a large dataset of peptide-binding affinities to HLA-DRB1*0101. The results showed that GAPES was reliable and very accurate. We achieved an average prediction accuracy of 93.50% and an average AUC of 0.974 in the IEDB dataset. Also, we achieved an accuracy of 95.125% and an AUC of 0.987 on the HLA-DRB1*0101 allele of the Wang benchmark dataset. The results indicate that the proposed prediction technique "GAPES" is a promising technique that will help researchers and scientists to predict the protein structure and it will assist them in the intelligent design of new epitope-based vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence for Globally Shared, Cross-Reacting Polymorphic Epitopes in the Pregnancy-Associated Malaria Vaccine Candidate VAR2CSA▿

PubMed Central

Avril, Marion; Kulasekara, Bridget R.; Gose, Severin O.; Rowe, Chris; Dahlbäck, Madeleine; Duffy, Patrick E.; Fried, Michal; Salanti, Ali; Misher, Lynda; Narum, David L.; Smith, Joseph D.

2008-01-01

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size (∼350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines. PMID:18250177

Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA.

PubMed

Avril, Marion; Kulasekara, Bridget R; Gose, Severin O; Rowe, Chris; Dahlbäck, Madeleine; Duffy, Patrick E; Fried, Michal; Salanti, Ali; Misher, Lynda; Narum, David L; Smith, Joseph D

2008-04-01

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

Antibody Epitope Analysis to Investigate Folded Structure, Allosteric Conformation, and Evolutionary Lineage of Proteins.

PubMed

Wong, Sienna; Jin, J-P

2017-01-01

Study of folded structure of proteins provides insights into their biological functions, conformational dynamics and molecular evolution. Current methods of elucidating folded structure of proteins are laborious, low-throughput, and constrained by various limitations. Arising from these methods is the need for a sensitive, quantitative, rapid and high-throughput method not only analysing the folded structure of proteins, but also to monitor dynamic changes under physiological or experimental conditions. In this focused review, we outline the foundation and limitations of current protein structure-determination methods prior to discussing the advantages of an emerging antibody epitope analysis for applications in structural, conformational and evolutionary studies of proteins. We discuss the application of this method using representative examples in monitoring allosteric conformation of regulatory proteins and the determination of the evolutionary lineage of related proteins and protein isoforms. The versatility of the method described herein is validated by the ability to modulate a variety of assay parameters to meet the needs of the user in order to monitor protein conformation. Furthermore, the assay has been used to clarify the lineage of troponin isoforms beyond what has been depicted by sequence homology alone, demonstrating the nonlinear evolutionary relationship between primary structure and tertiary structure of proteins. The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

PubMed

Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

2015-01-01

Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

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

[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.

Vicilin allergens of peanut and tree nuts (walnut, hazelnut and cashew nut) share structurally related IgE-binding epitopes.

PubMed

Barre, Annick; Sordet, Camille; Culerrier, Raphaël; Rancé, Fabienne; Didier, Alain; Rougé, Pierre

2008-03-01

Surface-exposed IgE-binding epitopes of close overall conformation were characterized on the molecular surface of three-dimensional models built for the vicilin allergens of peanut (Ara h 1), walnut (Jug r 2), hazelnut (Cor a 11) and cashew nut (Ana o 1). They correspond to linear stretches of conserved amino acid sequences mainly located along the C-terminus of the polypeptide chains. A glyco-epitope corresponding to an exposed N-glycosylation site could also interfere with the IgE-binding epitopes. All these epitopic regions should participate in the IgE-binding cross-reactivity commonly reported between tree nuts or between peanut and some tree nuts in sensitized individuals. Owing to this epitopic community which constitutes a risk of cross-sensitization, the avoidance or a restricted consumption of other tree nuts should be recommended to peanut-sensitized individuals.

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

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

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

PubMed Central

Pelle, Roger; Graham, Simon P.; Njahira, Moses N.; Osaso, Julius; Saya, Rosemary M.; Odongo, David O.; Toye, Philip G.; Spooner, Paul R.; Musoke, Anthony J.; Mwangi, Duncan M.; Taracha, Evans L. N.; Morrison, W. Ivan; Weir, William; Silva, Joana C.; Bishop, Richard P.

2011-01-01

Background Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8+ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8+ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8+ T-cell epitopes, and to analyse the sequences for evidence of selection. Methodology/Principal Findings Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. Conclusions/Significance The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point. PMID:21559495

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

PubMed

Pelle, Roger; Graham, Simon P; Njahira, Moses N; Osaso, Julius; Saya, Rosemary M; Odongo, David O; Toye, Philip G; Spooner, Paul R; Musoke, Anthony J; Mwangi, Duncan M; Taracha, Evans L N; Morrison, W Ivan; Weir, William; Silva, Joana C; Bishop, Richard P

2011-04-29

Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Sparse networks of directly coupled, polymorphic, and functional side chains in allosteric proteins.

PubMed

Soltan Ghoraie, Laleh; Burkowski, Forbes; Zhu, Mu

2015-03-01

Recent studies have highlighted the role of coupled side-chain fluctuations alone in the allosteric behavior of proteins. Moreover, examination of X-ray crystallography data has recently revealed new information about the prevalence of alternate side-chain conformations (conformational polymorphism), and attempts have been made to uncover the hidden alternate conformations from X-ray data. Hence, new computational approaches are required that consider the polymorphic nature of the side chains, and incorporate the effects of this phenomenon in the study of information transmission and functional interactions of residues in a molecule. These studies can provide a more accurate understanding of the allosteric behavior. In this article, we first present a novel approach to generate an ensemble of conformations and an efficient computational method to extract direct couplings of side chains in allosteric proteins, and provide sparse network representations of the couplings. We take the side-chain conformational polymorphism into account, and show that by studying the intrinsic dynamics of an inactive structure, we are able to construct a network of functionally crucial residues. Second, we show that the proposed method is capable of providing a magnified view of the coupled and conformationally polymorphic residues. This model reveals couplings between the alternate conformations of a coupled residue pair. To the best of our knowledge, this is the first computational method for extracting networks of side chains' alternate conformations. Such networks help in providing a detailed image of side-chain dynamics in functionally important and conformationally polymorphic sites, such as binding and/or allosteric sites. © 2014 Wiley Periodicals, Inc.

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

PubMed

Rotunno, Melissa S; Auclair, Jared R; Maniatis, Stephanie; Shaffer, Scott A; Agar, Jeffrey; Bosco, Daryl A

2014-10-10

Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp(92) and Asp(96) as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

DOE PAGES

Chen, Edwin; Salinas, Nichole D.; Huang, Yining; ...

2016-05-18

Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

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

Chen, Edwin; Salinas, Nichole D.; Huang, Yining

Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

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.

Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope

PubMed Central

Upadhyay, Chitra; Mayr, Luzia M.; Zhang, Jing; Kumar, Rajnish; Gorny, Miroslaw K.; Nádas, Arthur; Zolla-Pazner, Susan

2014-01-01

ABSTRACT Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4β7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (MAbs) display extensive cross-reactivity with gp120 monomers from many subtypes but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the neutralization of pseudoviruses produced in the presence of a glycosidase inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i MAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-MAb interaction to 18 h before adding target cells increased virus neutralization by some V2i MAbs and all V3 MAbs tested. Consistent with this, V2i MAb binding to Env on the surface of transfected cells also increased in a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites. IMPORTANCE Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking of V3 epitopes and V2i epitopes in the V1V2 domain. Importantly, V3 MAbs and some V2i MAbs display greater neutralization against relatively resistant HIV-1 isolates when the MAbs interact with the virus for a prolonged period of time. Given their highly immunogenic nature, V3 and V2i epitopes are valuable targets that would augment the efficacy of HIV vaccines. PMID:25165106

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

[Development and application of CK-MB specific monoclonal antibodies].

PubMed

Chen, Zimin; Zhou, Guoliang; Xu, Weiling; Zheng, Xiaohong; Tong, Xunzhang; Ke, Qishen; Song, Liuwei; Ge, Shengxiang

2017-01-25

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

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

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.

Enhancing antibody patent protection using epitope mapping information

PubMed Central

Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J.

2018-01-01

ABSTRACT As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents. PMID:29120697

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.

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.

Epitope-cavities generated by molecularly imprinted films measure the coincident response to anthrax protective antigen and its segments.

PubMed

Tai, Dar-Fu; Jhang, Ming-Hong; Chen, Guan-Yu; Wang, Sue-Chen; Lu, Kuo-Hao; Lee, Yu-Der; Liu, Hsin-Tzu

2010-03-15

A molecularly imprinted film was fabricated, in the presence of epitope-peptides, onto a quartz crystal microbalance (QCM) chip. These five peptides are known linear or conformational epitopes of the anthrax protective antigen PA(83). Imprinting resulted in an epitope-cavity with affinity for the corresponding template. With the use of a basic monomer, the binding-effect was further enhanced increasing the affinity to nanomolar levels. The affinities of the peptide to their corresponding molecularly induced polymers (MIPs) were more closely related to the molecular weight of the analyte than to the number of residues. All epitope-cavities differentiated their epitope region on the protective antigen PA(83) as well as the corresponding furin cleavage fragments PA(63) and PA(20). The QCM chip differential response to the protective antigen fragment was observed in the picomolar range, thus demonstrating a method to manipulate protein on the surface with defined orientation.

Shear, heat and pH induced conformational changes of wheat gluten - Impact on antigenicity.

PubMed

Rahaman, Toheder; Vasiljevic, Todor; Ramchandran, Lata

2016-04-01

Processing can induce conformational changes of food proteins depending on the conditions used that may affect their antigenicity. This study investigated the effect of pH (3,5,7) temperature (80,90,100 °C) and shear (500,1000,1500 s(-1)) on the conformational changes (surface hydrophobicity, FTIR, SDS-PAGE and thiol content) of gluten in relation to its antigenicity (determined by Enzyme-linked Immunosorbent Assay). Overall, at pH 3, up to 90 °C, conformational changes and possible burial of some antigenic hydrophobic residues resulted in reduction of antigenicity to one-third that of control. Further heating to 100 °C caused increase in antigenicity due to exposure of some hidden epitopes. However, at pH 5 and 7, the antigenicity declined only at 100 °C due to modification in thiol content and related structural changes causing destruction and/or masking of some epitopes. Shear alone had no effect on antigenicity of gluten but could have a synergistic influence at pH 7 and 100 °C. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

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.

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

The antigenic identity of human class I MHC phosphopeptides is critically dependent upon phosphorylation status

PubMed Central

Zarling, Angela L.; Willcox, Carrie R.; Shabanowitz, Jeffrey; Cummings, Kara L.; Hunt, Donald F.; Cobbold, Mark; Engelhard, Victor H.; Willcox, Benjamin E.

2017-01-01

Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches. PMID:28903331

Improved Method for Linear B-Cell Epitope Prediction Using Antigen’s Primary Sequence

PubMed Central

Raghava, Gajendra P. S.

2013-01-01

One of the major challenges in designing a peptide-based vaccine is the identification of antigenic regions in an antigen that can stimulate B-cell’s response, also called B-cell epitopes. In the past, several methods have been developed for the prediction of conformational and linear (or continuous) B-cell epitopes. However, the existing methods for predicting linear B-cell epitopes are far from perfection. In this study, an attempt has been made to develop an improved method for predicting linear B-cell epitopes. We have retrieved experimentally validated B-cell epitopes as well as non B-cell epitopes from Immune Epitope Database and derived two types of datasets called Lbtope_Variable and Lbtope_Fixed length datasets. The Lbtope_Variable dataset contains 14876 B-cell epitope and 23321 non-epitopes of variable length where as Lbtope_Fixed length dataset contains 12063 B-cell epitopes and 20589 non-epitopes of fixed length. We also evaluated the performance of models on above datasets after removing highly identical peptides from the datasets. In addition, we have derived third dataset Lbtope_Confirm having 1042 epitopes and 1795 non-epitopes where each epitope or non-epitope has been experimentally validated in at least two studies. A number of models have been developed to discriminate epitopes and non-epitopes using different machine-learning techniques like Support Vector Machine, and K-Nearest Neighbor. We achieved accuracy from ∼54% to 86% using diverse s features like binary profile, dipeptide composition, AAP (amino acid pair) profile. In this study, for the first time experimentally validated non B-cell epitopes have been used for developing method for predicting linear B-cell epitopes. In previous studies, random peptides have been used as non B-cell epitopes. In order to provide service to scientific community, a web server LBtope has been developed for predicting and designing B-cell epitopes (http://crdd.osdd.net/raghava/lbtope/). PMID:23667458

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

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.

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike

DOE PAGES

Cai, Yongfei; Karaca-Griffin, Selen; Chen, Jia; ...

2017-04-10

Here, the extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160) 3, cleaved to (gp120/gp41) 3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies.more » The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160) 3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.« less

Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike

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

Cai, Yongfei; Karaca-Griffin, Selen; Chen, Jia

Here, the extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160) 3, cleaved to (gp120/gp41) 3] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies.more » The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160) 3, retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens.« less

Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin

PubMed Central

Abdulaev, Najmoutin G.; Ridge, Kevin D.

1998-01-01

A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka ≈ 6 × 10−9 M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304–311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen–antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling. PMID:9789004

Minor Viral and Host Genetic Polymorphisms Can Dramatically Impact the Biologic Outcome of an Epitope-Specific CD8 T-Cell Response

DTIC Science & Technology

2009-08-20

be associated with impaired antigen process - ing.45,46 Indeed, extra-epitopic mutations were observed in B81 subjects infected with subtype C in whom...Medical Research Council (United Kingdom) Senior Clinical Fellow. Sample collection was supported by the European Commission, DG XII, INCO -DC (grant...Gall S, Pfafferott KJ, et al. Im- mune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J Exp

Thermal, spectroscopic, and ab initio structural characterization of carprofen polymorphs.

PubMed

Bruni, Giovanna; Gozzo, Fabia; Capsoni, Doretta; Bini, Marcella; Macchi, Piero; Simoncic, Petra; Berbenni, Vittorio; Milanese, Chiara; Girella, Alessandro; Ferrari, Stefania; Marini, Amedeo

2011-06-01

Commercial and recrystallized polycrystalline samples of carprofen, a nonsteroidal anti-inflammatory drug, were studied by thermal, spectroscopic, and structural techniques. Our investigations demonstrated that recrystallized sample, stable at room temperature (RT), is a single polymorphic form of carprofen (polymorph I) that undergoes an isostructural polymorphic transformation by heating (polymorph II). Polymorph II remains then metastable at ambient conditions. Commercial sample is instead a mixture of polymorphs I and II. The thermodynamic relationships between the two polymorphs were determined through the construction of an energy/temperature diagram. The ab initio structural determination performed on synchrotron X-Ray powder diffraction patterns recorded at RT on both polymorphs allowed us to elucidate, for the first time, their crystal structure. Both crystallize in the monoclinic space group type P2(1) /c, and the unit cell similarity index and the volumetric isostructurality index indicate that the temperature-induced polymorphic transformation I → II is isostructural. Polymorphs I and II are conformational polymorphs, sharing a very similar hydrogen bond network, but with different conformation of the propanoic skeleton, which produces two different packing. The small conformational change agrees with the low value of transition enthalpy obtained by differential scanning calorimetry measurements and the small internal energy computed with density functional methods. Copyright © 2011 Wiley-Liss, Inc.

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

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.

Anagostic interactions in chiral separation. Polymorphism in a [Co(II)(L)] complex: Crystallographic and theoretical studies

NASA Astrophysics Data System (ADS)

Awwadi, Firas F.; Hodali, Hamdallah A.

2018-02-01

Syntheses and crystal structures of two polymorphs of the complex [Co(II)(L)], where H2L = 2,2'-[cis-1,2-diaminocyclohexanediylbis (nitrilo-methylidyne)]bis (5-dimethyl-amino]phenol, have been studied. The two polymorphs concomitantly crystallized by vapour diffusion of solvent. The first polymorph (I) crystallized as a racemate in the centrosymmetric tetragonal I41/a space group. The second polymorph (II) crystallized in the chiral orthorhombic space group P212121. The chiral conformers of symmetrical cis-1,2-disubstituted cyclohexane molecules cannot be resolved in the liquid or gas phases, due to the rapid ring inversion. In the present study, the two chiral conformers are present in crystals of polymorph I, whereas, only one chiral conformer is present in crystals of polymorph II. Crystal structure analysis indicated that the formation of two different polymorphs of [Co(II)(L)] complex can be rationalized based on Csbnd H⋯Co anagostic interactions. Density Functional Theory (DFT) calculations indicated that Csbnd H⋯Co interactions are due to HOMO-LUMO interactions.

Crystalline structure of the marketed form of Rifampicin: a case of conformational and charge transfer polymorphism

NASA Astrophysics Data System (ADS)

de Pinho Pessoa Nogueira, Luciana; de Oliveira, Yara S.; de C. Fonseca, Jéssica; Costa, Wendell S.; Raffin, Fernanda N.; Ellena, Javier; Ayala, Alejandro Pedro

2018-03-01

Rifampicin is a semi-synthetic drug derived from rifamycin B, and currently integrates the fixed dose combination tablet formulations used in the treatment of tuberculosis. It is also used in the leprosy polychemotherapy and prophylaxis, which are diseases classified as neglected according to the World Health Organization. Rifampicin is a polymorphic drug and its desirable polymorphic form is labeled as II, being the main goal of this study the elucidation of its crystalline structure. Polymorph II is characterized by two molecules with different conformations in the asymmetric unit and the following lattice parameters: a = 14.0760 (10) Å, b = 17.5450 (10) Å, c = 17.5270 (10) Å, β = 92.15°. Differently to the previously reported structures, a charge transference from the hydroxyl group of the naphthoquinone of one conformer to the nitrogen of the piperazine group of the second conformer was observed. The relevance of the knowledge of this crystalline structure, which is the preferred polymorph for pharmaceutical formulations, was evidenced by analyzing raw materials with polymorphic mixtures. Thus, the results presented in this contribution close an old information gap allowing the complete solid-state characterization of rifampicin.

Polymorphism in liver-stage malaria vaccine candidate proteins: immune evasion and implications for vaccine design.

PubMed

Flanagan, Katie L; Wilson, Kirsty L; Plebanski, Magdalena

2016-01-01

The pre-erythrocytic stage of infection by malaria parasites represents a key target for vaccines that aim to eradicate malaria. Two important broad immune evasion strategies that can interfere with vaccine efficacy include the induction of dendritic cell (DC) dysfunction and regulatory T cells (Tregs) by blood-stage malaria parasites, leading to inefficient priming of T cells targeting liver-stage infections. The parasite also uses 'surgical strike' strategies, whereby polymorphism in pre-erythrocytic antigens can interfere with host immunity. Specifically, we review how even single amino acid changes in T cell epitopes can lead to loss of binding to major histocompatibility complex (MHC), lack of cross-reactivity, or antagonism and immune interference, where simultaneous or sequential stimulation with related variants of the same T cell epitope can cause T cell anergy or the conversion of effector to immunosuppressive T cell phenotypes.

A mathematical framework for the selection of an optimal set of peptides for epitope-based vaccines.

PubMed

Toussaint, Nora C; Dönnes, Pierre; Kohlbacher, Oliver

2008-12-01

Epitope-based vaccines (EVs) have a wide range of applications: from therapeutic to prophylactic approaches, from infectious diseases to cancer. The development of an EV is based on the knowledge of target-specific antigens from which immunogenic peptides, so-called epitopes, are derived. Such epitopes form the key components of the EV. Due to regulatory, economic, and practical concerns the number of epitopes that can be included in an EV is limited. Furthermore, as the major histocompatibility complex (MHC) binding these epitopes is highly polymorphic, every patient possesses a set of MHC class I and class II molecules of differing specificities. A peptide combination effective for one person can thus be completely ineffective for another. This renders the optimal selection of these epitopes an important and interesting optimization problem. In this work we present a mathematical framework based on integer linear programming (ILP) that allows the formulation of various flavors of the vaccine design problem and the efficient identification of optimal sets of epitopes. Out of a user-defined set of predicted or experimentally determined epitopes, the framework selects the set with the maximum likelihood of eliciting a broad and potent immune response. Our ILP approach allows an elegant and flexible formulation of numerous variants of the EV design problem. In order to demonstrate this, we show how common immunological requirements for a good EV (e.g., coverage of epitopes from each antigen, coverage of all MHC alleles in a set, or avoidance of epitopes with high mutation rates) can be translated into constraints or modifications of the objective function within the ILP framework. An implementation of the algorithm outperforms a simple greedy strategy as well as a previously suggested evolutionary algorithm and has runtimes on the order of seconds for typical problem sizes.

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.

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

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

Structural Basis of HCV Neutralization by Human Monoclonal Antibodies Resistant to Viral Neutralization Escape

PubMed Central

Krey, Thomas; Meola, Annalisa; Keck, Zhen-yong; Damier-Piolle, Laurence; Foung, Steven K. H.; Rey, Felix A.

2013-01-01

The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434–446 and aa610–619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434–446. The structures show that the peptide adopts an α-helical conformation with the main contact residues F442 and Y443 forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development. PMID:23696737

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

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

PubMed

Kumar, Vikram; Damodharan, S; Pandaranayaka, Eswari P J; Madathiparambil, Madanan G; Tennyson, Jebasingh

2016-01-01

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.

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

Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

PubMed Central

Falk, R J; Becker, M; Terrell, R; Jennette, J C

1992-01-01

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1379133

Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins.

PubMed

Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M H; Idrees, Muhammad

2016-12-09

The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV.

Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

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

McLellan, Jason S.; Correia, Bruno E.; Chen, Man

2012-06-28

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potentmore » neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.« less

Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus.

PubMed

McLellan, Jason S; Correia, Bruno E; Chen, Man; Yang, Yongping; Graham, Barney S; Schief, William R; Kwong, Peter D

2011-06-24

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required. Published by Elsevier Ltd.

LKM1 autoantibodies in chronic hepatitis C infection: a case of molecular mimicry?

PubMed

Marceau, Gabriel; Lapierre, Pascal; Béland, Kathie; Soudeyns, Hugo; Alvarez, Fernando

2005-09-01

Anti-liver-kidney microsome type 1 (LKM1) autoantibodies directed against the cytochrome P450 2D6 (CYP2D6) are considered specific markers of type 2 autoimmune hepatitis, but are also found in 5% of sera from patients chronically infected by hepatitis C virus (HCV). Molecular mimicry between HCV proteins and CYP2D6 has been proposed to explain the emergence of these autoantibodies. Anti-LKM1 autoantibodies from hepatitis C-infected patients were affinity-purified against immobilized CYP2D6 protein and used to screen a phage display library. CYP2D6 conformational epitopes were identified using phage display analysis and the identification of statistically significant pairs (SSPs). Cross-reactivity between CYP2D6 and HCV protein candidates was tested by immunoprecipitation. Nineteen different clones were isolated, and their sequencing resulted in the mapping of a conformational epitope to the region of amino acids 254-288 of CYP2D6. Candidate HCV proteins for molecular mimicry included: core, E2, NS3 and NS5a. Affinity-purified autoantibodies from HCV+/LKM1+ patients immunoprecipitated either NS3, NS5a, or both, and these reactivities were specifically inhibited by immobilized CYP2D6. In conclusion, HCV+/LKM1+ sera recognize a specific conformational epitope on CYP2D6 between amino acids 254 to 288, the region that contains the major linear epitope in type 2 autoimmune hepatitis patients. Cross-reactivity due to molecular mimicry at the B-cell level was shown between the CYP2D6 and the HCV NS3 and NS5a proteins and could explain the presence of anti-LKM1 in patients chronically infected with HCV. Further investigation of the role played by this molecular mimicry in HCV-infected patients may lead to more specific strategies for diagnosis and treatment.

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

PubMed Central

Fodor, James; Riley, Blake; Godlewska, Marlena; Góra, Monika; Czarnocka, Barbara; Banga, J Paul; Hoke, David E.; Kass, Itamar; Buckle, Ashley M.

2015-01-01

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data. PMID:26623656

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity.

PubMed

Le, Sarah N; Porebski, Benjamin T; McCoey, Julia; Fodor, James; Riley, Blake; Godlewska, Marlena; Góra, Monika; Czarnocka, Barbara; Banga, J Paul; Hoke, David E; Kass, Itamar; Buckle, Ashley M

2015-01-01

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.

Epitope mapping: the first step in developing epitope-based vaccines.

PubMed

Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael

2007-01-01

Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have been developed, such as Mapitope, which has recently been found to be effective in mapping conformational discontinuous epitopes. The pros and cons of various approaches towards epitope mapping are also discussed.

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

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

Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

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

Li Jing; Beijing Key Laboratory for Protein Therapeutics, Beijing 100084; Chen Xi

2008-11-07

The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformationmore » as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.« less

Presence of the p.L456V polymorphism in Cuban patients clinically diagnosed with Wilson's disease.

PubMed

Clark-Feoktistova, Y; Ruenes-Domech, C; García-Bacallao, E F; Roblejo-Balbuena, H; Feoktistova, L; Clark-Feoktistova, I; Jay-Herrera, O; Collazo-Mesa, T

2018-06-10

Wilson's disease is characterized by the accumulation of copper in different organs, mainly affecting the liver, brain, and cornea, and is caused by mutations in the ATP7B gene. More than 120 polymorphisms in the ATP7B gene have been reported in the medical literature. The aim of the present study was to identify the conformational changes in the exon 3 region of the ATP7B gene and detect the p.L456V polymorphism in Cuban patients clinically diagnosed with Wilson's disease. A descriptive study was conducted at the Centro Nacional de Genética Médica and the Instituto Nacional de Gastroenterología within the time frame of 2007-2012 and included 105 patients with a clinical diagnosis of Wilson's disease. DNA extraction was performed through the salting-out method and the fragment of interest was amplified using the polymerase chain reaction technique. The conformational shift changes in the exon 3 region and the presence of the p.L456V polymorphism were identified through the Single-Strand Conformation Polymorphism analysis. The so-called b and c conformational shift changes, corresponding to the p.L456V polymorphism in the heterozygous and homozygous states, respectively, were identified. The allelic frequency of the p.L456V polymorphism in the 105 Cuban patients that had a clinical diagnosis of Wilson's disease was 41% and liver-related symptoms were the most frequent in the patients with that polymorphism. The p.L456V polymorphism was identified in 64 Cuban patients clinically diagnosed with Wilson's disease, making future molecular study through indirect methods possible. Copyright © 2018 Asociación Mexicana de Gastroenterología. Publicado por Masson Doyma México S.A. All rights reserved.

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

Two polymorphs of 2,5-dichloro-3,6-bis(dibenzylamino)-p-hydroquinone with flexible dibenzylamino groups.

PubMed

Shin, In Sub; Shimada, Yuta; Horiguchi-Babamoto, Emi; Matsumoto, Shinya

2018-04-01

We obtained two conformational polymorphs of 2,5-dichloro-3,6-bis(dibenzylamino)-p-hydroquinone, C 34 H 30 Cl 2 N 2 O 2 . Both polymorphs have an inversion centre at the centre of the hydroquinone ring (Z' = 1/2), and there are no significant differences between their bond lengths and angles. The most significant structural difference in the molecular conformations was found in the rotation of the phenyl rings of the two crystallographically independent benzyl groups. The crystal structures of the polymorphs were distinguishable with respect to the arrangement of the hydroquinone rings and the packing motif of the phenyl rings that form part of the benzyl groups. The phenyl groups of one polymorph are arranged in a face-to-edge motif between adjacent molecules, with intermolecular C-H...π interactions, whereas the phenyl rings in the other polymorph form a lamellar stacking pattern with no significant intermolecular interactions. We suggest that this partial conformational difference in the molecular structures leads to the significant structural differences observed in their molecular arrangements.

Polymorphs and polymorphic cocrystals of temozolomide.

PubMed

Babu, N Jagadeesh; Reddy, L Sreenivas; Aitipamula, Srinivasulu; Nangia, Ashwini

2008-07-07

Crystal polymorphism in the antitumor drug temozolomide (TMZ), cocrystals of TMZ with 4,4'-bipyridine-N,N'-dioxide (BPNO), and solid-state stability were studied. Apart from a known X-ray crystal structure of TMZ (form 1), two new crystalline modifications, forms 2 and 3, were obtained during attempted cocrystallization with carbamazepine and 3-hydroxypyridine-N-oxide. Conformers A and B of the drug molecule are stabilized by intramolecular amide N--HN(imidazole) and N--HN(tetrazine) interactions. The stable conformer A is present in forms 1 and 2, whereas both conformers crystallized in form 3. Preparation of polymorphic cocrystals I and II (TMZBPNO 1:0.5 and 2:1) were optimized by using solution crystallization and grinding methods. The metastable nature of polymorph 2 and cocrystal II is ascribed to unused hydrogen-bond donors/acceptors in the crystal structure. The intramolecularly bonded amide N-H donor in the less stable structure makes additional intermolecular bonds with the tetrazine C==O group and the imidazole N atom in stable polymorph 1 and cocrystal I, respectively. All available hydrogen-bond donors and acceptors are used to make intermolecular hydrogen bonds in the stable crystalline form. Synthon polymorphism and crystal stability are discussed in terms of hydrogen-bond reorganization.

Xenoepitope substitution avoids deceptive imprinting and broadens the immune response to foot-and-mouth disease virus.

PubMed

Szczepanek, Steven M; Barrette, Roger W; Rood, Debra; Alejo, Diana; Silbart, Lawrence K

2012-04-01

Many RNA viruses encode error-prone polymerases which introduce mutations into B and T cell epitopes, providing a mechanism for immunological escape. When regions of hypervariability are found within immunodominant epitopes with no known function, they are referred to as "decoy epitopes," which often deceptively imprint the host's immune response. In this work, a decoy epitope was identified in the foot-and-mouth disease virus (FMDV) serotype O VP1 G-H loop after multiple sequence alignment of 118 isolates. A series of chimeric cyclic peptides resembling the type O G-H loop were prepared, each bearing a defined "B cell xenoepitope" from another virus in place of the native decoy epitope. These sequences were derived from porcine respiratory and reproductive syndrome virus (PRRSV), from HIV, or from a presumptively tolerogenic sequence from murine albumin and were subsequently used as immunogens in BALB/c mice. Cross-reactive antibody responses against all peptides were compared to a wild-type peptide and ovalbumin (OVA). A broadened antibody response was generated in animals inoculated with the PRRSV chimeric peptide, in which virus binding of serum antibodies was also observed. A B cell epitope mapping experiment did not reveal recognition of any contiguous linear epitopes, raising the possibility that the refocused response was directed to a conformational epitope. Taken together, these results indicate that xenoepitope substitution is a novel method for immune refocusing against decoy epitopes of RNA viruses such as FMDV as part of the rational design of next-generation vaccines.

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.

Role of C-Terminal Cysteine Residues of Aspergillus fumigatus Allergen Asp f 4 in Immunoglobulin E Binding

PubMed Central

Ramachandran, Harikrishnan; Banerjee, Banani; Greenberger, Paul A.; Kelly, Kevin J.; Fink, Jordan N.; Kurup, Viswanath P.

2004-01-01

Among the several allergens cloned and expressed from Aspergillus fumigatus, Asp f 4 is a major one associated with allergic bronchopulmonary aspergillosis (ABPA). The structure-function relationship of allergens is important in understanding the immunopathogenesis, diagnosis, and treatment of allergic diseases. These include the epitopes, conformational or linear, deletion of the N or C terminus or both N and C termini, and glycosylation or nonglycosylation, all of which affect immune responses. Similarly, the role of cysteine residues present in allergens may yield useful information regarding the conformational structure of allergens and the immunoglobulin E (IgE) epitope interaction. Such information may help in developing new strategies towards immunotherapy. In order to define the role of cysteine in the interaction of the antibody with Asp f 4, we have constructed mutants by selectively deleting cysteine residues from the C-terminal region of the Asp f 4. Immunological evaluation of these engineered recombinant constructs was conducted by using sera from patients with ABPA, Aspergillus skin test-positive asthmatics, and healthy controls. The results demonstrate strong IgE binding with Asp f 4 and two truncated mutants, Asp f 41-234 (amino acids [aa] 1 to 234) and Asp f 41-241 (aa 1 to 241), while another mutant, Asp f 41-196 (aa 1 to 196), showed reactivity with fewer patients. The result suggests that deletion of cysteines and the alteration of IgE epitopes at the C-terminal end resulted in conformational changes, which may have a potential role in the immunomodulation of the disease. PMID:15013973

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.

Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

PubMed

Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

2016-06-01

Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1

PubMed Central

Brumme, Chanson J.; Martin, Eric; Listgarten, Jennifer; Brockman, Mark A.; Le, Anh Q.; Chui, Celia K. S.; Cotton, Laura A.; Knapp, David J. H. F.; Riddler, Sharon A.; Haubrich, Richard; Nelson, George; Pfeifer, Nico; DeZiel, Charles E.; Heckerman, David; Apps, Richard; Carrington, Mary; Mallal, Simon; Harrigan, P. Richard; John, Mina

2012-01-01

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV's structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies. PMID:23055555

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

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.

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.

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.

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

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Non-rigid molecule of copper(II) diiminate Cu[CF3C(NH)C(F)C(NH)CF3]2, its conformational polymorphism in crystal and structure in solutions (Raman, UV-vis and quantum chemistry study)

NASA Astrophysics Data System (ADS)

Bukalov, Sergey S.; Aysin, Rinat R.; Leites, Larissa A.; Kurykin, Mikhail A.; Khrustalev, Victor N.

2015-10-01

Calculation of potential energy surface (PES) of isolated molecule of copper(II) diiminate Cu[CF3С(NH)C(F)C(NH)CF3]2 (1) resulted a double-well curve with the minima corresponding to equivalent screwed conformations. The low barrier leads to molecular non-rigidity which seems to be the reason of conformational polymorphism in crystals, reported in [1]. For one of newly found polymorphs, the X-ray structure was determined. The differences in the Raman and UV-vis spectra between differently colored species and their solutions were revealed, they are determined by different geometries of Cu(II) coordination polyhedron and different systems of intermolecular interactions in crystals. Transformations of the polymorphs under thermal, mechanical and photo exposures were studied.

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

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

Pierce, Brian G.; Boucher, Elisabeth N.; Piepenbrink, Kurt H.

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, asmore » well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCEHepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies.In vivoresults in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus.« less

Characterization of single chain antibody targets through yeast two hybrid

PubMed Central

2010-01-01

Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine. PMID:20727208

Persistent hydrogen bonding in polymorphic crystal structures.

PubMed

Galek, Peter T A; Fábián, László; Allen, Frank H

2009-02-01

The significance of hydrogen bonding and its variability in polymorphic crystal structures is explored using new automated structural analysis methods. The concept of a chemically equivalent hydrogen bond is defined, which may be identified in pairs of structures, revealing those types of bonds that may persist, or not, in moving from one polymorphic form to another. Their frequency and nature are investigated in 882 polymorphic structures from the Cambridge Structural Database. A new method to compare conformations of equivalent molecules is introduced and applied to derive distinct subsets of conformational and packing polymorphs. The roles of chemical functionality and hydrogen-bond geometry in persistent interactions are systematically explored. Detailed structural comparisons reveal a large majority of persistent hydrogen bonds that are energetically crucial to structural stability.

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

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

[High frequency of ancestral allele of the TJP1 polymorphism rs2291166 in Mexican population, conformational effect and applications in surgery and medicine].

PubMed

Ramirez-Garcia, Sergio Alberto; Flores-Alvarado, Luis Javier; Topete-González, Luz Rosalba; Charles-Niño, Claudia; Mazariegos-Rubi, Manuel; Dávalos-Rodríguez, Nory Omayra

2016-01-01

TJP1 gene encodes a ZO-1 protein that is required for the recruitment of occludins and claudins in tight junction, and is involved in cell polarisation. It has different variations, the frequency of which has been studied in different populations. In Mexico there are no studies of this gene. These are required because their polymorphisms can be used in studies associated with medicine and surgery. Therefore, the aim of this study was to estimate the frequency of alleles and genotypes of rs2291166 gene polymorphism TJP1 in Mexico Mestizos population, and to estimate the conformational effect of an amino acid change. A total of 473 individuals were included. The rs2291166 polymorphism was identified PASA PCR-7% PAGE, and stained with silver nitrate. The conformational effect of amino acid change was performed in silico, and was carried out with servers ProtPraram Tool and Search Database with Fasta. The most frequent allele in the two populations is the ancestral allele (T). A genotype distribution similar to other populations was found. The polymorphism is in Hardy-Weinberg, p>0.05. Changing aspartate to alanine produced a conformational change. The study reveals a high frequency of the ancestral allele at rs2291166 polymorphism in the Mexican population. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

Conformational polymorphism and thermochemical analysis of 5,5' ''-bis[(2,2,5,5-tetramethyl-1-aza-2,5-disila-1-cyclopentyl)ethyl]-2,2':5',2' ':5' ',2' ''-quaterthiophene.

PubMed

Muguruma, Hitoshi; Hotta, Shu

2006-11-23

The titled compound exists as two polymorphic solid phases (denoted form-I and form-II). Form-I obtained by as-synthesized material is a more stable phase. Form-II is a less stable phase. Spontaneous solid-solid transformation from form-II to form-I is observed in the temperature range between room temperature and the melting point of form-I (Tm = 156.5 degrees C), and its activation energy is estimated to be 96 kJ mol-1 by Arrhenius plot. The solid-solute-solid transformation (recrystallization from solution) from form-II to form-I is also observed. In contrast, form-II is obtained only by a solid-melt-solid transformation from form-I. Therefore, the system of two polymorphs is monotropic. The solid-state NMR measurement shows that form-I has the molecular conformation of complete S-syn-anti-syn in the oligothiophene backbone, whereas form-II has that of S-all-anti. With the solution NMR data, the polymorphism could not be observed. Therefore, the polymorphs originate from the different molecular packing involving the conformational change of the molecule. This unique property is attributed to the extra bulky terminal groups of the compounds. However, despite the extra bulky terminal groups, the mentioned polymorphism is not observed in the titled compound analogue which has S-all-anti conformation (like form-II).

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

Conformational Changes of Blood ACE in Chronic Uremia

PubMed Central

Petrov, Maxim N.; Shilo, Valery Y.; Tarasov, Alexandr V.; Schwartz, David E.; Garcia, Joe G. N.; Kost, Olga A.; Danilov, Sergei M.

2012-01-01

Background The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. Hypothesis Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. Methodology/Principal Findings The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The “short” ACE inhibitor enalaprilat (tripeptide analog) and “long” inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. Conclusions/Significance The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy. PMID:23166630

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.

Identification, Characterization, and Epitope Mapping of Human Monoclonal Antibody J19 That Specifically Recognizes Activated Integrin α4β7*

PubMed Central

Qi, JunPeng; Zhang, Kun; Zhang, Qiao; Sun, Yi; Fu, Ting; Li, GuoHui; Chen, JianFeng

2012-01-01

Integrin α4β7 is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α4β7, which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α4β7. Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α4β7. J19 was discovered by screening a human single-chain variable fragment phage library using an activated α4β7 mutant as target. J19 IgG specifically bound to the high affinity α4β7 induced by Mn2+, DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α4β7-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of β7 I domain and a seven-residue segment from 184 to 190 of α4 β-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α4β7 activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α4β7. PMID:22418441

Uncommon Pathways of Immune Escape Attenuate HIV-1 Integrase Replication Capacity

PubMed Central

Chopera, Denis R.; Olvera, Alex; Brumme, Chanson J.; Sela, Jennifer; Markle, Tristan J.; Martin, Eric; Carlson, Jonathan M.; Le, Anh Q.; McGovern, Rachel; Cheung, Peter K.; Kelleher, Anthony D.; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P. Richard; Heckerman, David; Brander, Christian; Walker, Bruce D.; Brumme, Zabrina L.

2012-01-01

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies. PMID:22496233

Uncommon pathways of immune escape attenuate HIV-1 integrase replication capacity.

PubMed

Brockman, Mark A; Chopera, Denis R; Olvera, Alex; Brumme, Chanson J; Sela, Jennifer; Markle, Tristan J; Martin, Eric; Carlson, Jonathan M; Le, Anh Q; McGovern, Rachel; Cheung, Peter K; Kelleher, Anthony D; Jessen, Heiko; Markowitz, Martin; Rosenberg, Eric; Frahm, Nicole; Sanchez, Jorge; Mallal, Simon; John, Mina; Harrigan, P Richard; Heckerman, David; Brander, Christian; Walker, Bruce D; Brumme, Zabrina L

2012-06-01

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = -0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF(114-121)) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ~35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.

Single-strand conformation polymorphism analysis of ribosomal DNA for detection of Phytophthora ramorum directly from plant tissues

Treesearch

Ping Kong; Patricia A. Richardson; Chuanxue Hong; Thomas L. Kubisiak

2006-01-01

At the first Sudden Oak Death Science Symposium, we reported on the use of a single strand conformation polymorphism (SSCP) analysis for rapid identification of Phytophthora ramorum in culture. We have since assessed and improved the fingerprinting technique for detecting this pathogen directly from plant tissues. The improved SSCP protocol uses a...

Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta

PubMed Central

Vasilevko, Vitaly; Pop, Viorela; Kim, Hyun Jin; Saing, Tommy; Glabe, Charles C.; Milton, Saskia; Barrett, Edward G.; Cotman, Carl W.; Cribbs, David H.; Head, Elizabeth

2010-01-01

Previously we showed that anti-Aβ peptide immunotherapy significantly attenuated Alzheimer’s-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4 years in canines immunized repeatedly with aggregated Aβ1–42 (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Aβ antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Aβ1–42 peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Aβ and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings. PMID:20451612

Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA.

PubMed

Dans, Pablo Daniel; Faustino, Ignacio; Battistini, Federica; Zakrzewska, Krystyna; Lavery, Richard; Orozco, Modesto

2014-10-01

We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3'-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175.

PubMed

Ambroggio, Xavier; Jiang, Lubin; Aebig, Joan; Obiakor, Harold; Lukszo, Jan; Narum, David L

2013-01-01

The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines.

PubMed

Lockyer, Kay; Gao, Fang; Derrick, Jeremy P; Bolgiano, Barbara

2015-03-10

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

PubMed Central

Lockyer, Kay; Gao, Fang; Derrick, Jeremy P.; Bolgiano, Barbara

2015-01-01

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8 × 106 g/mol to larger than 20 × 106 g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. PMID:25640334

Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

PubMed

Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

2008-07-22

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody

PubMed Central

Vulliez-Le Normand, B.; Saul, F. A.; Phalipon, A.; Bélot, F.; Guerreiro, C.; Mulard, L. A.; Bentley, G. A.

2008-01-01

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes. PMID:18621718

Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies

PubMed Central

Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

2015-01-01

Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage. PMID:26605008

Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies.

PubMed

Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

2015-01-01

Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage.

A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

PubMed Central

Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

2013-01-01

1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization. PMID:24278299

Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

PubMed Central

Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

2016-01-01

Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

PubMed Central

Gurung, Ratna B.; Purdie, Auriol C.; Begg, Douglas J.

2012-01-01

Johne's disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts. PMID:22496492

Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease.

PubMed

Gianfrani, Carmen; Mamone, Gianfranco; la Gatta, Barbara; Camarca, Alessandra; Di Stasio, Luigia; Maurano, Francesco; Picascia, Stefania; Capozzi, Vito; Perna, Giuseppe; Picariello, Gianluca; Di Luccia, Aldo

2017-03-01

Microwave based treatment (MWT) of wet wheat kernels induced a striking reduction of gluten, up to <20 ppm as determined by R5-antibodybased ELISA, so that wheat could be labeled as gluten-free. In contrast, analysis of gluten peptides by G12 antibody-based ELISA, mass spectrometry-based proteomics and in vitro assay with T cells of celiac subjects, indicated no difference of antigenicity before and after MWT. SDS-PAGE analysis and Raman spectroscopy demonstrated that MWT simply induced conformational modifications, reducing alcohol solubility of gliadins and altering the access of R5-antibody to the gluten epitopes. Thus, MWT neither destroys gluten nor modifies chemically the toxic epitopes, contradicting the preliminary claims that MWT of wheat kernels detoxifies gluten. This study provides evidence that R5-antibody ELISA alone is not effective to determine gluten in thermally treated wheat products. Gluten epitopes in processed wheat should be monitored using strategies based on combined immunoassays with T cells from celiacs, G12-antibody ELISA after proteolysis and proper molecular characterization. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses.

PubMed

Richard, Jonathan; Prévost, Jérémie; Baxter, Amy E; von Bredow, Benjamin; Ding, Shilei; Medjahed, Halima; Delgado, Gloria G; Brassard, Nathalie; Stürzel, Christina M; Kirchhoff, Frank; Hahn, Beatrice H; Parsons, Matthew S; Kaufmann, Daniel E; Evans, David T; Finzi, Andrés

2018-03-20

The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a "closed" conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands. IMPORTANCE Emerging evidence supports a role for antibody-dependent cellular cytotoxicity (ADCC) in protection against HIV-1 transmission and disease progression. However, there are conflicting reports regarding the ability of nonneutralizing antibodies targeting CD4-inducible (CD4i) Env epitopes to mediate ADCC. Here, we performed a side-by-side comparison of different methods currently being used in the field to measure ADCC responses to HIV-1. We found that assays which are unable to differentiate virus-infected from uninfected cells greatly overestimate ADCC responses mediated by antibodies to CD4i epitopes and underestimate responses mediated by broadly neutralizing antibodies (bNAbs). Our results strongly argue for the use of assays that measure ADCC against HIV-1-infected cells expressing physiologically relevant conformations of Env to evaluate correlates of protection in vaccine trials. Copyright © 2018 Richard et al.

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.

Polymorphism in the nitrate salt of the [Mn(acetylacetonate)2(H2O)2]+ ion.

PubMed

Biju, A R; Rajasekharan, M V

2010-06-01

The crystallization of [Mn(acac)(2)(H(2)O)(2)](+) from solutions containing excess nitrate leads to the formation of four polymorphs. All polymorphs contain two different types of complex ions, one containing essentially coplanar acac ligands and the other in which the two acac ligands together assume a chair conformation. Molecular modelling using DFT (density-functional theory) calculations shows that the coplanar conformation is the electronically stable one. The hydrogen bonding between the trans-water molecules and the nitrate ion produces a one-dimensional chain of 12-membered rings, which are further organized into a two-dimensional network via a lattice water molecule. Lattice-energy calculations have been carried out to compare the stabilities of the four polymorphs.

Polymorphism at 129 dictates metastable conformations of the human prion protein N-terminal β-sheet† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc03275c Click here for additional data file.

PubMed Central

Paz, S. Alexis; Vanden-Eijnden, Eric

2017-01-01

We study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly parameterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V, D178N) polymorphs have a ground-state conformation with three intact β-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion “strain” hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation. PMID:28451263

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

Decreased immunoglobulin E (IgE) binding to cashew allergens following sodium sulfite treatment and heating

USDA-ARS?s Scientific Manuscript database

Cashew nut and other nut allergies can result in serious and sometimes life threatening reactions. Linear and conformational epitopes within food allergens are important for immunoglobulin E binding. Methods that disrupt allergen structure can reduce immunoglobulin E binding and lessen the likelih...

Identification of a Polymorphic Gene, BCL2A1, Encoding Two Novel Hematopoietic Lineage-specific Minor Histocompatibility Antigens

PubMed Central

Akatsuka, Yoshiki; Nishida, Tetsuya; Kondo, Eisei; Miyazaki, Mikinori; Taji, Hirohumi; Iida, Hiroatsu; Tsujimura, Kunio; Yazaki, Makoto; Naoe, Tomoki; Morishima, Yasuo; Kodera, Yoshihisa; Kuzushima, Kiyotaka; Takahashi, Toshitada

2003-01-01

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1. PMID:12771180

Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail

PubMed Central

Pelle, Roger; Mwacharo, Joram M.; Njahira, Moses N.; Marcellino, Wani L.; Kiara, Henry; Malak, Agol K.; EL Hussein, Abdel Rahim M.; Bishop, Richard; Skilton, Robert A.

2017-01-01

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail. PMID:28231338

Structural analysis of the RH-like blood group gene products in nonhuman primates

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

Salvignol, I.; Calvas, P.; Blancher, A.

1995-03-01

Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M{sub r},more » immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh-like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene. 55 refs., 4 figs., 1 tab.« less

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

PubMed

Lee, Augustine S; Finkielman, Javier D; Peikert, Tobias; Hummel, Amber M; Viss, Margaret A; Specks, Ulrich

2005-12-20

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.

Antigenic Structure of the Human Muscle Nicotinic Acetylcholine Receptor Main Immunogenic Region

PubMed Central

Luo, Jie; Lindstrom, Jon

2009-01-01

The main immunogenic region on the α1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation-dependence of its epitopes by making chimeras in which sequences of human muscle α1 subunits were replaced in human neuronal α7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation, and, consequently, assembly of receptor subunits. PMID:19705087

Concomitant and conformational polymorphism in 4‧-(isoquinolyl-2,2‧:6‧,2″-terpyridine and 4‧-(4-quinolyl)-2,2‧:6‧,2″-terpyridine

NASA Astrophysics Data System (ADS)

Njogu, Eric M.; Nyamori, Vincent O.; Omondi, Bernard

2018-02-01

The occurrence of concomitant polymorphism in 4‧-(isoquinolyl)-2,2‧:6‧,2″-terpyridine, 1a and 1b (2-quinterpy) and conformational polymorphism in 4‧-(4-quinolyl)-2,2‧:6‧,2″-terpyridine (4-quinterpy) has been identified to due to crystallization process and solvent, respectively. Crystallization of 2-quinterpy in acetone yielded the concomitant polymorphs 1a and 1b which crystallize in the monoclinic P21/c and the orthorhombic Pna21 space groups, respectively. The polymorph 2a was grown from bulk 4-quinterpy in dimethyl sulfoxide, crystallizes in the monoclinic P21/c space group, while 2b grown from acetonitrile or even acetone crystallizes in the monoclinic system but in P21/n space group.

Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

PubMed

Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

2012-01-01

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

USDA-ARS?s Scientific Manuscript database

Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8positive T-cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presenta...

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

PubMed Central

Nicholls, Sarah; Piper, Karen P.; Mohammed, Fiyaz; Dafforn, Timothy R.; Tenzer, Stefan; Salim, Mahboob; Mahendra, Premini; Craddock, Charles; van Endert, Peter; Schild, Hansjörg; Cobbold, Mark; Engelhard, Victor H.; Moss, Paul A. H.; Willcox, Benjamin E.

2009-01-01

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1H versus HA-1R) in the HMHA1 gene. The HA-1H peptide is restricted by HLA-A2 and is immunogenic in HA-1R/R into HA-1H transplants, while HA-1R has been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1R variant. To understand these findings, we determined the structure of an HLA-A2-HA-1H complex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1H peptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition. PMID:19234124

Antigenic Fingerprinting following Primary RSV Infection in Young Children Identifies Novel Antigenic Sites and Reveals Unlinked Evolution of Human Antibody Repertoires to Fusion and Attachment Glycoproteins

PubMed Central

Fuentes, Sandra; Coyle, Elizabeth M.; Beeler, Judy; Golding, Hana; Khurana, Surender

2016-01-01

Respiratory Syncytial Virus (RSV) is the major cause of pneumonia among infants. Here we elucidated the antibody repertoire following primary RSV infection and traced its evolution through adolescence and adulthood. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes in the RSV fusion protein (F) and attachment protein (G) were used for unbiased epitope profiling of infant sera prior to and following RSV infection. F-GFPDL analyses demonstrated modest changes in the anti-F epitope repertoires post-RSV infection, while G-GFPDL analyses revealed 100-fold increase in number of bound phages. The G-reactive epitopes spanned the N- and C-terminus of the G ectodomain, along with increased reactivity to the central conserved domain (CCD). Panels of F and G antigenic sites were synthesized to evaluate sera from young children (<2 yr), adolescents (14–18 yr) and adults (30–45 yr) in SPR real-time kinetics assays. A steady increase in RSV-F epitope repertoires from young children to adults was observed using peptides and F proteins. Importantly, several novel epitopes were identified in pre-fusion F and an immunodominant epitope in the F-p27. In all age groups, antibody binding to pre-fusion F was 2–3 folds higher than to post-fusion form. For RSV-G, antibody responses were high following early RSV infection in children, but declined significantly in adults, using either G proteins or peptides. This study identified unlinked evolution of anti-F and anti G responses and supportive evidence for immune pressure driven evolution of RSV-G. These findings could help development of effective countermeasures including vaccines. PMID:27100289

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

Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

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

Kanai,R.; Kar, K.; Anthony, K.

2006-01-01

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specificmore » antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.« less

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

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy

PubMed Central

Walls, Alexandra C.; Tortorici, M. Alejandra; Frenz, Brandon; Snijder, Joost; Li, Wentao; Rey, Félix A.; DiMaio, Frank; Bosch, Berend-Jan; Veesler, David

2017-01-01

The threat of a major coronavirus pandemic urges the development of suitable strategies to combat these pathogens. HCoV-NL63 is an α-coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. We report here the 3.4 Å resolution cryo-electron microscopy reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass-spectrometry, provides a structural framework to understand accessibility to antibodies. The structure also reveals a remarkable modular architecture of the receptor-binding subunit and the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on masking of epitopes with glycans and activating conformational changes, to evade the immune system of infected hosts. PMID:27617430

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

A paradigm shift: Cancer therapy with peptide-based B-cell epitopes and peptide immunotherapeutics targeting multiple solid tumor types: Emerging concepts and validation of combination immunotherapy

PubMed Central

Kaumaya, Pravin TP

2015-01-01

There is a recognizable and urgent need to speed the development and application of novel, more efficacious anti-cancer vaccine therapies that inhibit tumor progression and prevent acquisition of tumor resistance. We have created and established a portfolio of validated peptide epitopes against multiple receptor tyrosine kinases and we have identified the most biologically effective combinations of EGFR (HER-1), HER-2, HER-3, VEGF and IGF-1R peptide vaccines/mimics to selectively inhibit multiple receptors and signaling pathways. The strategy is based on the use of chimeric conformational B-cell epitope peptides incorporating “promiscuous” T-cell epitopes that afford the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide antibodies as potential vaccines and peptide mimics that act as antagonists to receptor signaling that drive cancer metastasis. In this review we will summarize our ongoing studies based on the development of combinatorial immunotherapeutic strategies that act synergistically to enhance immune-mediated tumor killing aimed at addressing mechanisms of tumor resistance for several tumor types. PMID:25874884

Chimaera and its modern virus-like descendants.

PubMed

Ulrich, R; Gerlich, W H; Krüger, D H

1996-01-01

Chimaera was a monster in ancient Greek mythology combining elements from different animal species in its body. Modern molecular biology enabled the generation of harmless but useful chimaeras consisting of elements from different nonrelated viruses. The objective is that the resulting chimaeras form highly immunogenic virus-like particles (VLPs). Such chimaeric VLPs can be used as highly efficient carriers for sequential and conformational B cell epitopes and T cell epitopes. Most VLPs are readily produced in heterologous hosts and are easy to purify. This article deals with various systems of VLPs described in this topical issue of Intervirology and makes comparisons with chimaeric replication-competent viruses, recombinant virus vectors expressing foreign proteins, and DNA vaccines.

Methods and Protocols for Developing Prion Vaccines.

PubMed

Marciniuk, Kristen; Taschuk, Ryan; Napper, Scott

2016-01-01

Prion diseases denote a distinct form of infectivity that is based in the misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Efforts to develop vaccines for prion diseases have been complicated by the potential dangers that are associated with induction of immune responses against a self-protein. As a consequence, there is considerable appeal for vaccines that specifically target the misfolded prion conformation. Such conformation-specific immunotherapy is made possible through the identification of vaccine targets (epitopes) that are exclusively presented as a consequence of misfolding. An immune response directed against these targets, termed disease-specific epitopes (DSEs), has the potential to spare the function of the native form of the protein while clearing, or neutralizing, the infectious isomer. Although identification of DSEs represents a critical first step in the induction of conformation-specific immune responses, substantial efforts are required to translate these targets into functional vaccines. Due to the poor immunogenicity that is inherent to self-proteins, and that is often associated with short peptides, substantial efforts are required to overcome tolerance-to-self and maximize the resultant immune response following DSE-based immunization. This often includes optimization of target sequences in terms of immunogenicity and development of effective formulation and delivery strategies for the associated peptides. Further, these vaccines must satisfy additional criteria from perspectives of specificity (PrP(C) vs. PrP(Sc)) and safety (antibody-induced template-driven misfolding of PrP(C)). The emphasis of this report is on the steps required to translate DSEs into prion vaccines and subsequent evaluation of the resulting immune responses.

PADI4 and the HLA-DRB1 shared epitope in juvenile idiopathic arthritis.

PubMed

Hisa, Kaori; Yanagimachi, Masakatsu D; Naruto, Takuya; Miyamae, Takako; Kikuchi, Masako; Hara, Rhoki; Imagawa, Tomoyuki; Yokota, Shumpei; Mori, Masaaki

2017-01-01

Both genetic and environmental factors are associated with susceptibility to juvenile idiopathic arthritis (JIA). Many studies have reported that both a 'shared epitope' (SE) encoded by several HLA-DRB1 alleles and the peptidyl arginine deiminase type 4 (PADI4) gene polymorphisms are associated with susceptibility to rheumatoid arthritis (RA). However, it is uncertain whether JIA and RA share the latter genetic risk factor. Therefore, here we investigated relationships between HLA-SE and PADI4 polymorphisms with clinical subtypes of JIA. JIA patients (39 oligoarthritis, 48 RF-positive polyarthritis, 19 RF-negative polyarthritis and 82 systemic) and 188 healthy controls were genotyped for HLA-DRB1 by PCR-sequence-specific oligonucleotide probe methodology. Three PADI4 gene single nucleotide polymorphisms (SNPs), rs2240340, rs2240337 and rs1748033, were genotyped using TaqMan SNP Genotyping Assays. Frequencies of the HLA-SE were higher in RF-positive polyarticular JIA than in healthy controls. RF-positive polyarticular JIA was associated with HLA-SE (OR = 5.3, 95% CI = 2.5-11.9, pc < 0.001). No associations were found between clinical subtypes of JIA and PADI4 allele frequency. Nonetheless, rs2240337 in the PADI4 gene was significantly associated with anti-cyclic citrullinated peptide antibody (ACPA)-positivity in JIA. The A allele at rs2240337 was a significant risk factor for ACPA positivity in JIA (OR = 5.6, 95% CI = 1.71-23.7 pc = 0.03). PADI4 gene polymorphism is associated with ACPA-positivity in JIA. The association of HLA-SE with RF-positive polyarticular JIA as well as RA is confirmed in Japanese. Thus, HLA-SE and PADI4 status both influence JIA clinical manifestations.

Polymorphism at codon 36 of the p53 gene.

PubMed

Felix, C A; Brown, D L; Mitsudomi, T; Ikagaki, N; Wong, A; Wasserman, R; Womer, R B; Biegel, J A

1994-01-01

A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

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

The Extracellular Domain of Myelin Oligodendrocyte Glycoprotein Elicits Atypical Experimental Autoimmune Encephalomyelitis in Rat and Macaque Species

PubMed Central

Curtis, Alan D.; Taslim, Najla; Reece, Shaun P.; Grebenciucova, Elena; Ray, Richard H.; Rosenbaum, Matthew D.; Wardle, Robert L.; Van Scott, Michael R.; Mannie, Mark D.

2014-01-01

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction. PMID:25303101

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

PubMed

Curtis, Alan D; Taslim, Najla; Reece, Shaun P; Grebenciucova, Elena; Ray, Richard H; Rosenbaum, Matthew D; Wardle, Robert L; Van Scott, Michael R; Mannie, Mark D

2014-01-01

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund's adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund's adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6-7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

PubMed Central

Petrova, Yuliya I.; Spano, MarthaJoy M.; Gumbiner, Barry M.

2012-01-01

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin–catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor–induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane. PMID:22513089

Characterization of the glycoprotein of infectious hematopoietic necrosis virus using neutralizing monoclonal antibodies

USGS Publications Warehouse

Huang, Chienjin; Chien, Maw-Sheng; Landolt, Marsha; Winton, James

1994-01-01

To study the antigenic nature of the glycoprotein (G protein) of infectious hematopoietic necrosis virus (IHNV), 31 neutralizing monoclonal antibodies (MAbs) were produced against a reference isolate of the virus. The MAbs were compared using a neutralization assay, an enzyme-linked immunosorbent assay (ELISA), and by immunoblotting of the G protein in the native, reduced, and deglycosylated forms. Hybridoma culture fluids of the various MAbs could be diluted from 1:2 to 1:512 and still completely neutralize 1 X 104 plaque-forming units of IHNV. Similarly, the end point dilutions that produced optical density readings of 0.1 or greater in the ELISA were 1:40 to 1:10240. Western blotting showed that all of the MAbs reacted with the G protein in the unreduced (i.e. native) conformation; however, only 9 nine of the MAbs were able to react with the G protein following reduction by 2-mercaptoethanol. Deglycosylation of the protein did not influence the binding ability of any of the MAbs. These data indicate that all the MAbs recognized amino acid sequences on the protein itself and that the IHNV glycoprotein contains linear as well as conformation-dependent neutralizing epitopes. When rainbow trout Oncorhynchus mykiss fingerlings were passively immunized with MAbs against either a linear or a conformation-dependent epitope, the fish were protected against challenge with wild-type IHNV.

Neutralization tiers of HIV-1

PubMed Central

Montefiori, David C.; Roederer, Mario; Morris, Lynn; Seaman, Michael S.

2018-01-01

Purpose of review HIV-1 isolates are often classified on the basis of neutralization ‘tier’ phenotype. Tier classification has important implications for the monitoring and interpretation of vaccine-elicited neutralizing antibody responses. The molecular basis that distinguishes the multiple neutralization phenotypes of HIV-1 has been unclear. We present a model based on the dynamic nature of the HIV-1 envelope glycoproteins and its impact on epitope exposure. We also describe a new approach for ranking HIV-1 vaccine-elicited neutralizing antibody responses. Recent findings The unliganded trimeric HIV-1 envelope glycoprotein spike spontaneously transitions through at least three conformations. Neutralization tier phenotypes correspond to the frequency by which the trimer exists in a closed (tiers 2 and 3), open (tier 1A), or intermediate (tier 1B) conformation. An increasing number of epitopes become exposed as the trimer opens, making the virus more sensitive to neutralization by certain antibodies. The closed conformation is stabilized by many broadly neutralizing antibodies. Summary The tier 2 neutralization phenotype is typical of most circulating strains and is associated with a predominantly closed Env trimer configuration that is a high priority to target with vaccines. Assays with tier 1A viruses should be interpreted with caution and with the understanding that they detect many antibody specificities that do not neutralize tier 2 viruses and do not protect against HIV-1 infection. PMID:29266013

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.

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.

A molecular dynamics study of loop fluctuation in human papillomavirus type 16 virus-like particles: a possible indicator of immunogenicity.

PubMed

Joshi, Harshad; Cheluvaraja, Srinath; Somogyi, Endre; Brown, Darron R; Ortoleva, Peter

2011-11-28

Immunogenicity varies between the human papillomavirus (HPV) L1 monomer assemblies of various sizes (e.g., monomers, pentamers or whole capsids). The hypothesis that this can be attributed to the intensity of fluctuations of important loops containing neutralizing epitopes for the various assemblies is proposed for HPV L1 assemblies. Molecular dynamics simulations were utilized to begin testing this hypothesis. Fluctuations of loops that contain critical neutralizing epitopes (especially FG loop) were quantified via root-mean-square fluctuation and features in the frequency spectrum of dynamic changes in loop conformation. If this fluctuation-immunogenicity hypothesis is a universal aspect of immunogenicity (i.e., immune system recognition of an epitope within a loop is more reliable when it is presented via a more stable delivery structure), then fluctuation measures can serve as one predictor of immunogenicity as part of a computer-aided vaccine design strategy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Design of three-component vaccines against group A streptococcal infections: importance of spatial arrangement of vaccine components.

PubMed

Abdel-Aal, Abu-Baker M; Zaman, Mehfuz; Fujita, Yoshio; Batzloff, Michael R; Good, Michael F; Toth, Istvan

2010-11-25

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4(+) T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.

A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens

PubMed Central

Goldblum, Randall M.; Ning, Bo; Judy, Barbara. M.; Holthauzen, Luis Marcelo F.; van Bavel, Julius; Kamijo, Atsushi; Midoro-Horiuti, Terumi

2016-01-01

We recently described a dominant role for conformational epitopes on the group 1 allergen of the mountain cedar (Juniperus ashei, Cupressaceae), Jun a 1, in pollen hypersensitivity in South Central U.S.A. Since these epitopes are surface exposed and are likely to be flexible, they may be susceptible to molecular or physical perturbations. This may make Jun a 1 a potential target for new forms of therapy for cedar pollinosis. Here, we describe a mouse monoclonal antibody, termed E58, which binds to the group 1 allergens of cedar pollens from three highly populated regions of the world (central U.S.A., France and Japan). Upon binding to these allergens, E58 strongly reduces the binding of patient’s IgE antibodies to these dominant allergens. This characteristic of E58, and potentially other similar antibodies, suggests an opportunity to identify preventative or therapeutic agents that may inhibit cedar pollen sensitization or prevent the allergic reactions. PMID:27174188

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

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.

PubMed

Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R; Rao, Sridhar; Jobaliya, Chintan; Poncz, Mortimer; French, Deborah L; Newman, Peter J

2016-02-11

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use. © 2016 by The American Society of Hematology.

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes

PubMed Central

Zhang, Nanyan; Zhi, Huiying; Curtis, Brian R.; Rao, Sridhar; Jobaliya, Chintan; Poncz, Mortimer; French, Deborah L.

2016-01-01

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the PlA1/PlA2 polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33+ megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41+ megakaryocyte progenitors derived from these cells expressed the HPA-1b (PlA2) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b–specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use. PMID:26634302

The interplay between the paracetamol polymorphism and its molecular structures dissolved in supercritical CO2 in contact with the solid phase: In situ vibration spectroscopy and molecular dynamics simulation analysis.

PubMed

Oparin, Roman D; Moreau, Myriam; De Walle, Isabelle; Paolantoni, Marco; Idrissi, Abdenacer; Kiselev, Michael G

2015-09-18

The aim of this paper is to characterize the distribution of paracetamol conformers which are dissolved in a supercritical CO2 phase being in equilibrium with their corresponding crystalline form. The quantum calculations and molecular dynamics simulations were used in order to characterize the structure and analyze the vibration spectra of the paracetamol conformers in vacuum and in a mixture with CO2 at various thermodynamic state parameters (p,T). The metadynamics approach was applied to efficiently sample the various conformers of paracetamol. Furthermore, using in situ IR spectroscopy, the conformers that are dissolved in supercritical CO2 were identified and the evolution of the probability of their presence as a functions of thermodynamic condition was quantified while the change in the crystalline form of paracetamol have been monitored by DSC, micro IR and Raman techniques. The DSC analysis as well as micro IR and Raman spectroscopic studies of the crystalline paracetamol show that the subsequent heating up above the melting temperature of the polymorph I of paracetamol and the cooling down to room temperature in the presence of supercritical CO2 induces the formation of polymorph II. The in situ IR investigation shows that two conformers (Conf. 1 and Conf. 2) are present in the phase of CO2 while conformer 3 (Conf. 3) has a high probability to be present after re-crystallization. Copyright © 2015. Published by Elsevier B.V.

[Association of muscle segment homeobox gene 1 polymorphisms with nonsyndromic cleft lip with or without cleft palate].

PubMed

Zhang, Li; Tang, Jun-Ling; Liang, Shang-Zheng

2008-06-01

Muscle segment homeobox gene (MSX)1 has been proposed as a gene in which mutations may contribute to nonsyndromic cleft lip with or without cleft palate (NSCL/P). To study MSX1 polymorphisms in NSCL/ P by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), and investigate the association of MSX1 exons 1 polymorphisms with NSCL/P. DNA were extracted from blood samples from NSCL/P and unrelated normal subjects. Genome DNA from peripheral leukocyte with these blood samples were extracted, which was used as template to amplify desired gene fragment of MSX1 exons 1 by means of polymerase chain reaction (PCR). The PCR products were examined by single-strand conformation polymorphism (SSCP). The MSX1 exons 1 polymorphisms were examined by sequencing if mutations were found. MSX1 genes of exon 1 mutation was not been found in the NSCL/P and unrelated normal subjects by SSCP. No correlation between MSX1 exon 1 and NSCL/P was found. MSX1 exon 1 may not be a key gene (susceptibility gene) in NSCL/P.

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.

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

Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies.

PubMed

Meola, Annalisa; Tarr, Alexander W; England, Patrick; Meredith, Luke W; McClure, C Patrick; Foung, Steven K H; McKeating, Jane A; Ball, Jonathan K; Rey, Felix A; Krey, Thomas

2015-02-01

Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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

Computational identification, characterization and validation of potential antigenic peptide vaccines from hrHPVs E6 proteins using immunoinformatics and computational systems biology approaches

PubMed Central

Junaid, Muhammad; Kaushik, Aman Chandra; Ali, Arif; Ali, Syed Shujait; Mehmood, Aamir; Wei, Dong-Qing

2018-01-01

High-risk human papillomaviruses (hrHPVs) are the most prevalent viruses in human diseases including cervical cancers. Expression of E6 protein has already been reported in cervical cancer cases, excluding normal tissues. Continuous expression of E6 protein is making it ideal to develop therapeutic vaccines against hrHPVs infection and cervical cancer. Therefore, we carried out a meta-analysis of multiple hrHPVs to predict the most potential prophylactic peptide vaccines. In this study, immunoinformatics approach was employed to predict antigenic epitopes of hrHPVs E6 proteins restricted to 12 Human HLAs to aid the development of peptide vaccines against hrHPVs. Conformational B-cell and CTL epitopes were predicted for hrHPVs E6 proteins using ElliPro and NetCTL. The potential of the predicted peptides were tested and validated by using systems biology approach considering experimental concentration. We also investigated the binding interactions of the antigenic CTL epitopes by using docking. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlighted the regions from 46–62 and 65–76 that could be the first choice for the development of prophylactic peptide vaccines against hrHPVs. To overcome the worldwide distribution, the predicted epitopes restricted to different HLAs could cover most of the vaccination and would help to explore the possibility of these epitopes for adaptive immunotherapy against HPVs infections. PMID:29715318

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.

PubMed

Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F M; Oliveira, Daysiane; Machado de Avila, Ricardo A; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S; Minozzo, João C; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

2018-01-01

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho , and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

PubMed Central

Lima, Sabrina de Almeida; Guerra-Duarte, Clara; Costal-Oliveira, Fernanda; Mendes, Thais Melo; Figueiredo, Luís F. M.; Oliveira, Daysiane; Machado de Avila, Ricardo A.; Ferrer, Valéria Pereira; Trevisan-Silva, Dilza; Veiga, Silvio S.; Minozzo, João C.; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

2018-01-01

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms. PMID:29666624

Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants.

PubMed

Dromey, James A; Weenink, Sarah M; Peters, Günther H; Endl, Josef; Tighe, Patrick J; Todd, Ian; Christie, Michael R

2004-04-01

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.

Conserved stem fragment from H3 influenza hemagglutinin elicits cross-clade neutralizing antibodies through stalk-targeted blocking of conformational change during membrane fusion.

PubMed

Gong, Xin; Yin, He; Shi, Yuhua; Guan, Shanshan; He, Xiaoqiu; Yang, Lan; Yu, Yongjiao; Kuai, Ziyu; Jiang, Chunlai; Kong, Wei; Wang, Song; Shan, Yaming

2016-04-01

Currently available influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies due to the mutability of virus sequences and conformational changes during protective immunity, thereby limiting their efficacy. This problem needs to be addressed by further understanding the mechanisms of neutralization and finding the desired neutralizing site during membrane fusion. This study specifically focused on viruses of the H3N2 subtype, which have persisted as a principal source of influenza-related morbidity and mortality in humans since the 1968 influenza pandemic. Through sequence alignment and epitope prediction, a series of highly conserved stem fragments (spanning 47 years) were found and coupled to the Keyhole Limpet Hemocyanin (KLH) protein. By application of a combinatorial display library and crystal structure modeling, a stem fragment immunogen, located at the turning point of the HA neck undergoing conformational change during membrane fusion with both B- and T-cell epitopes, was identified. After synthesis of the optimal stem fragment using a multiple antigen peptide (MAP) system, strong humoral immune responses and cross-clade neutralizing activities against strains from the H3 subtype of group 2 influenza viruses after animal immunizations were observed. By detection of nuclear protein immunofluorescence with acid bypass treatment, antisera raised against MAP4 immunogens of the stem fragment showed the potential to inhibit the conformational change of HA in stem-targeted virus neutralization. The identification of this conserved stem fragment provides great potential for exploitation of this site of vulnerability in therapeutic and vaccine design. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Controlled conformational transitions in the MVM virion expose the VP1 N-terminus and viral genome without particle disassembly.

PubMed

Cotmore, S F; D'abramo, A M; Ticknor, C M; Tattersall, P

1999-02-01

Antisera were raised against peptides corresponding to the N-termini of capsid proteins VP1 and VP2 from the parvovirus minute virus of mice. Epitopes in the 142-amino-acid VP1-specific region were not accessible in the great majority of newly released viral particles, and sera directed against them failed to neutralize virus directly or deplete stocks of infectious virions. However, brief exposure to temperatures of 45 degreesC or more induced a conformational transition in a population of full virions, but not in empty viral particles, in which VP1-specific sequences became externally accessible. In contrast, the VP2 N-terminus was antibody-accessible in all full, but not empty, particles without prior treatment. An electrophoretic mobility shift assay, in which particles were heat-treated and/or preincubated with antibodies prior to electrophoresis, confirmed this pattern of epitope accessibility, showing that the heat-induced conformational transition produces a retarded form of virion that can be supershifted by incubation with VP1-specific sera. The proportion of virions undergoing transition increased with temperature, but at all temperatures up to 70 degreesC viral particles retained structure-specific antigenic determinants and remained essentially intact, without shedding individual polypeptide species or subunits. However, despite the apparent integrity of its protective coat, the genome became accessible to externally applied enzymes in an increasing proportion of virions through this temperature range, suggesting that the conformational transitions that expose VP1 likely also allow access to the genome. Heating particles to 80 degreesC or above finally induced disassembly to polypeptide monomers. Copyright 1999 Academic Press.

Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo*

PubMed Central

García-Medel, Noel; Sanz-Bravo, Alejandro; Van Nguyen, Dung; Galocha, Begoña; Gómez-Molina, Patricia; Martín-Esteban, Adrián; Alvarez-Navarro, Carlos; de Castro, José A. López

2012-01-01

The association of ERAP1 with ankylosing spondylitis (AS)1 among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 interaction is a variant-dependent alteration in the balance between epitope generation and destruction determined by the susceptibility of N-terminal flanking and P1 residues to trimming. ERAP1 polymorphism associated with AS susceptibility ensured efficient peptide trimming and high HLA-B27 stability. Protective polymorphism resulted in diminished ERAP1 activity, less efficient trimming, suboptimal HLA-B27 peptidomes, and decreased molecular stability. This study demonstrates that natural ERAP1 polymorphism affects HLA-B27 antigen presentation and stability in vivo and proposes a mechanism for the interaction between these molecules in AS. PMID:22918227

Myelography Iodinated Contrast Media. 2. Conformational Versatility of Iopamidol in the Solid State.

PubMed

Bellich, Barbara; Di Fonzo, Silvia; Tavagnacco, Letizia; Paolantoni, Marco; Masciovecchio, Claudio; Bertolotti, Federica; Giannini, Giovanna; De Zorzi, Rita; Geremia, Silvano; Maiocchi, Alessandro; Uggeri, Fulvio; Masciocchi, Norberto; Cesàro, Attilio

2017-02-06

The phenomenon of polymorphism is of great relevance in pharmaceutics, since different polymorphs have different physicochemical properties, e.g., solubility, hence, bioavailability. Coupling diffractometric and spectroscopic experiments with thermodynamic analysis and computational work opens to a methodological approach which provides information on both structure and dynamics in the solid as well as in solution. The present work reports on the conformational changes in crystalline iopamidol, which is characterized by atropisomerism, a phenomenon that influences both the solution properties and the distinct crystal phases. The conformation of iopamidol is discussed for three different crystal phases. In the anhydrous and monohydrate crystal forms, iopamidol molecules display a syn conformation of the long branches stemming out from the triiodobenzene ring, while in the pentahydrate phase the anti conformation is found. IR and Raman spectroscopic studies carried out on the three crystal forms, jointly with quantum chemical computations, revealed that the markedly different spectral features can be specifically attributed to the different molecular conformations. Our results on the conformational versatility of iopamidol in different crystalline phases, linking structural and spectroscopic evidence for the solution state and the solid forms, provide a definite protocol for grasping the signals that can be taken as conformational markers. This is the first step for understanding the crystallization mechanism occurring in supersaturated solution of iopamidol molecules.

Transmitted/Founder HIV-1 Subtype C Viruses Show Distinctive Signature Patterns in Vif, Vpr, and Vpu That Are Under Subsequent Immune Pressure During Early Infection.

PubMed

Rossenkhan, Raabya; MacLeod, Iain J; Brumme, Zabrina L; Magaret, Craig A; Sebunya, Theresa K; Musonda, Rosemary; Gashe, Berhanu A; Edlefsen, Paul T; Novitsky, Vlad; Essex, M

Viral variants that predominate during early infection may exhibit constrained diversity compared with those found during chronic infection and could contain amino acid signature patterns that may enhance transmission, establish productive infection, and influence early events that modulate the infection course. We compared amino acid distributions in 17 patients recently infected with HIV-1C with patients with chronic infection. We found significantly lower entropy in inferred transmitted/founder (t/f) compared with chronic viruses and identified signature patterns in Vif and Vpr from inferred t/f viruses. We investigated sequence evolution longitudinally up to 500 days postseroconversion and compared the impact of selected substitutions on predicted human leukocyte antigen (HLA) binding affinities of published and predicted cytotoxic T-lymphocyte epitopes. Polymorphisms in Vif and Vpr during early infection occurred more frequently at epitope-HLA anchor residues and significantly decreased predicted epitope-HLA binding. Transmission-associated sequence signatures may have implications for novel strategies to prevent HIV-1 transmission.

Transmitted/Founder HIV-1 Subtype C Viruses Show Distinctive Signature Patterns in Vif, Vpr, and Vpu That Are Under Subsequent Immune Pressure During Early Infection

PubMed Central

Rossenkhan, Raabya; MacLeod, Iain J.; Brumme, Zabrina L.; Magaret, Craig A.; Sebunya, Theresa K.; Musonda, Rosemary; Gashe, Berhanu A.; Edlefsen, Paul T.; Novitsky, Vlad

2016-01-01

Abstract Viral variants that predominate during early infection may exhibit constrained diversity compared with those found during chronic infection and could contain amino acid signature patterns that may enhance transmission, establish productive infection, and influence early events that modulate the infection course. We compared amino acid distributions in 17 patients recently infected with HIV-1C with patients with chronic infection. We found significantly lower entropy in inferred transmitted/founder (t/f) compared with chronic viruses and identified signature patterns in Vif and Vpr from inferred t/f viruses. We investigated sequence evolution longitudinally up to 500 days postseroconversion and compared the impact of selected substitutions on predicted human leukocyte antigen (HLA) binding affinities of published and predicted cytotoxic T-lymphocyte epitopes. Polymorphisms in Vif and Vpr during early infection occurred more frequently at epitope-HLA anchor residues and significantly decreased predicted epitope-HLA binding. Transmission-associated sequence signatures may have implications for novel strategies to prevent HIV-1 transmission. PMID:27349335

DEVELOPMENT OF CODOMINANT MARKERS FOR IDENTIFYING SPECIES HYBRIDS

EPA Science Inventory

Herein we describe a simple method for developing species-diagnostic markers that would permit the rapid identification of hybrid individuals. Our method relies on amplified length polymorphism (AFLP) and single strand conformation polymorphism (SSCP) technologies, both of which...

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.

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.

Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis

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

Meyer, J.M.

The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR{beta} chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation {chi}{sup 2} method was used to evaluate evidence for linkagemore » disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal {chi}{sup 2}. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401, *0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women. 67 refs., 7 tabs.« less

Differentiation among isolates of prunus necrotic ringspot virus by transcript conformation polymorphism.

PubMed

Rosner, A; Maslenin, L; Spiegel, S

1998-09-01

A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.

Bonobos maintain immune-system diversity with three functional types of MHC-B1

PubMed Central

Wroblewski, Emily E.; Guethlein, Lisbeth A.; Norman, Paul J.; Li, Yingying; Shaw, Christiana M.; Han, Alex S.; Ndjango, Jean-Bosco N.; Ahuka-Mundeke, Steve; Georgiev, Alexander V.; Peeters, Martine; Hahn, Beatrice H.; Parham, Peter

2017-01-01

Fast-evolving MHC class I polymorphism serves to diversify NK cell and CD8 T cell responses in individuals, families, and populations. As only chimpanzee and bonobo have strict orthologs of all HLA class I, their study gives unique perspective on the human condition. We defined polymorphism of Papa-B, the bonobo ortholog of HLA-B, for six wild bonobo populations. Sequences for Papa-B exon 2 and 3 were determined from the genomic DNA in 255 fecal samples, minimally representing 110 individuals. Twenty-two Papa-B alleles were defined, each encoding a different Papa-B protein. No Papa-B is identical to any chimpanzee Patr-B, human HLA-B, or gorilla Gogo-B. Phylogenetic analysis identified a clade of MHC-B, defined by residues 45–74 of the α1 domain, which is broadly conserved among bonobo, chimpanzee, and gorilla. Bonobo populations have 3–14 Papa-B allotypes. Three Papa-B are in all populations, and they are each of a different functional type: allotypes having the Bw4 epitope recognized by killer cell immunoglobulin-like receptors (KIR) of NK cells, allotypes having the C1 epitope also recognized by KIR, and allotypes having neither epitope. For population ML these three Papa-B are the only Papa-B allotypes. Although small in number, their sequence divergence is such that the nucleotide diversity (mean p-distance) of Papa-B in ML is greater than in the other populations, and also greater than expected for random combinations of three Papa-B. Overall, Papa-B has substantially less diversity than Patr-B in chimpanzee subspecies and HLA-B in indigenous human populations, consistent with bonobo having experienced narrower population bottlenecks. PMID:28348269

Structural Effects of Two Camelid Nanobodies Directed to Distinct C-Terminal Epitopes on α-Synuclein.

PubMed

El-Turk, Farah; Newby, Francisco N; De Genst, Erwin; Guilliams, Tim; Sprules, Tara; Mittermaier, Anthony; Dobson, Christopher M; Vendruscolo, Michele

2016-06-07

α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.

Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics

NASA Astrophysics Data System (ADS)

Kordopati, Golfo G.; Tzoupis, Haralambos; Troganis, Anastassios N.; Tsivgoulis, Gerasimos M.; Golic Grdadolnik, Simona; Simal, Carmen; Tselios, Theodore V.

2017-09-01

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

Molecular Basis of the Behavior of Hepatitis A Virus Exposed to High Hydrostatic Pressure

PubMed Central

D'Andrea, Lucía; Pérez-Rodríguez, Francisco J.; Costafreda, M. Isabel; Beguiristain, Nerea; Fuentes, Cristina; Aymerich, Teresa; Guix, Susana; Bosch, Albert

2014-01-01

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis. PMID:25107980

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

Role of molecular mimicry to HIV-1 peptides in HIV-1–related immunologic thrombocytopenia

PubMed Central

Li, Zongdong; Nardi, Michael A.; Karpatkin, Simon

2005-01-01

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the β3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti–GPIIIa49-66–induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated. PMID:15774614

Narrow Groove and Restricted Anchors of MHC Class I Molecule BF2*0401 Plus Peptide Transporter Restriction can Explain Disease Susceptibility of B4 Chickens

PubMed Central

Zhang, Jianhua; Chen, Yong; Qi, Jianxun; Gao, Feng; Liu, Yanjie; Liu, Jun; Zhou, Xuyu; Kaufman, Jim; Xia, Chun; Gao, George F.

2016-01-01

The major histocompatibility complex (MHC) has genetic associations with many diseases, often due to differences in presentation of antigenic peptides by polymorphic MHC molecules to T lymphocytes of the immune system. In chickens, only a single classical class I molecule in each MHC haplotype is expressed well due to co-evolution with the polymorphic transporters associated with antigen presentation (TAPs), which means that resistance and susceptibility to infectious pathogens are particularly easy to observe. Previously, structures of chicken MHC class I molecule BF2*2101 from B21 haplotype showed an unusually large peptide-binding groove that accommodates a broad spectrum of peptides to present as epitopes to cytotoxic T lymphocytes (CTL), explaining the MHC-determined resistance of B21 chickens to Marek's disease. Here, we report the crystal structure of BF2*0401 from the B4 (also known as B13) haplotype, showing a highly positively-charged surface hitherto unobserved in other MHC molecules, as well as a remarkably narrow groove due to the allele-specific residues with bulky side chains. Together, these properties limit the number of epitope peptides that can bind this class I molecule. However, peptide-binding assays show that in vitro BF2*0401 can bind a wider variety of peptides than are found on the surface of B4 cells. Thus, a combination of the specificities of the polymorphic TAP transporter and the MHC results in a very limited set of BF2*0401 peptides with negatively charged anchors to be presented to T lymphocytes. PMID:23041567

Crystal structure of carnidazole form II from synchrotron X-ray powder diffraction: structural comparison with form I, the hydrated form and the low energy conformations in vacuo.

PubMed

de Armas, Héctor Novoa; Peeters, Oswald M; Blaton, Norbert; Van den Mooter, Guy; De Ridder, Dirk J A; Schenk, Henk

2006-10-01

The crystal structure of carnidazole form II, O-methyl [2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethyl]thiocarbamate, has been determined using synchrotron X-ray powder diffraction in combination with simulated annealing and whole profile pattern matching, and refined by the Rietveld method. For structure solution, 12 degrees of freedom were defined: one motion group and six torsions. Form II crystallizes in space group P2(1)/n, Z=4, with unit cell parameters after Rietveld refinement: a=13.915(4), b=8.095(2), c=10.649(3) A, beta=110.83(1) degrees, and V=1121.1(5) A3. The two polymorphic forms, as well as the hydrate, crystallize in the monoclinic space group P2(1)/n having four molecules in the cell. In form II, the molecules are held together by forming two infinite zig-zag chains via hydrogen bonds of the type N--H...N, the same pattern as in form I. A conformational study of carnidazole, at semiempirical PM3 level, was performed using stochastic approaches based on modification of the flexible torsion angles. The values of the torsion angles for the molecules of the two polymorphic forms and the hydrate of carnidazole are compared to those obtained from the conformational search. Form I and form II are enantiotropic polymorphic pairs this agrees with the fact that the two forms are conformational polymorphs. Copyright (c) 2006 Wiley-Liss, Inc. and the American Pharmacists Association

A Plasmodium falciparum 48/45 single epitope R0.6C subunit protein elicits high levels of transmission blocking antibodies.

PubMed

Singh, Susheel K; Roeffen, Will; Andersen, Gorm; Bousema, Teun; Christiansen, Michael; Sauerwein, Robert; Theisen, Michael

2015-04-15

The sexual stage Pfs48/45 antigen is a well-established lead candidate for a transmission blocking (TB) vaccine because of its critical role in parasite fertilization. We have recently produced the carboxy-terminal 10C-fragment of Pfs48/45 containing three known epitopes for TB antibodies as a chimera with the N-terminal region of GLURP (R0). The resulting fusion protein elicited high titer TB antibodies in rodents. To increase the relatively low yield of correctly folded Pfs48/45 we have generated a series of novel chimera truncating the 10C-fragments to 6 cysteine residues containing sub-units (6C). All constructs harbor the major epitope I for TB antibodies. One of these sub-units (R0.6Cc), produced high yields of correctly folded conformers, which could be purified by a simple 2-step procedure. Purified R0.6Cc was stable and elicits high titer TB antibodies in rats. The yield, purity and stability of R0.6Cc allows for further clinical development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

PubMed

Uchtenhagen, Hannes; Abualrous, Esam T; Stahl, Evi; Allerbring, Eva B; Sluijter, Marjolein; Zacharias, Martin; Sandalova, Tatyana; van Hall, Thorbald; Springer, Sebastian; Nygren, Per-Åke; Achour, Adnane

2013-11-01

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma.

PubMed

Hwang, Hai-Min; Heo, Chang-Kyu; Lee, Hye Jung; Kwak, Sang-Seob; Lim, Won-Hee; Yoo, Jong-Shin; Yu, Dae-Yuel; Lim, Kook Jin; Kim, Jeong-Yoon; Cho, Eun-Wie

2018-06-28

Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum. To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens. In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081). ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.

The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease.

PubMed

Bhattacharjee, Arnab; Reuter, Stefanie; Trojnár, Eszter; Kolodziejczyk, Robert; Seeberger, Harald; Hyvärinen, Satu; Uzonyi, Barbara; Szilágyi, Ágnes; Prohászka, Zoltán; Goldman, Adrian; Józsi, Mihály; Jokiranta, T Sakari

2015-04-10

Atypical hemolytic uremic syndrome (aHUS) is characterized by complement attack against host cells due to mutations in complement proteins or autoantibodies against complement factor H (CFH). It is unknown why nearly all patients with autoimmune aHUS lack CFHR1 (CFH-related protein-1). These patients have autoantibodies against CFH domains 19 and 20 (CFH19-20), which are nearly identical to CFHR1 domains 4 and 5 (CFHR14-5). Here, binding site mapping of autoantibodies from 17 patients using mutant CFH19-20 constructs revealed an autoantibody epitope cluster within a loop on domain 20, next to the two buried residues that are different in CFH19-20 and CFHR14-5. The crystal structure of CFHR14-5 revealed a difference in conformation of the autoantigenic loop in the C-terminal domains of CFH and CFHR1, explaining the variation in binding of autoantibodies from some aHUS patients to CFH19-20 and CFHR14-5. The autoantigenic loop on CFH seems to be generally flexible, as its conformation in previously published structures of CFH19-20 bound to the microbial protein OspE and a sialic acid glycan is somewhat altered. Cumulatively, our data suggest that association of CFHR1 deficiency with autoimmune aHUS could be due to the structural difference between CFHR1 and the autoantigenic CFH epitope, suggesting a novel explanation for CFHR1 deficiency in the pathogenesis of autoimmune aHUS. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses

PubMed Central

Richard, Jonathan; Prévost, Jérémie; Baxter, Amy E.; Ding, Shilei; Medjahed, Halima; Delgado, Gloria G.; Brassard, Nathalie; Stürzel, Christina M.; Kirchhoff, Frank; Hahn, Beatrice H.; Parsons, Matthew S.; Kaufmann, Daniel E.; Evans, David T.

2018-01-01

ABSTRACT The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a “closed” conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands. PMID:29559570

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies.

PubMed

Majumdar, Ritankar; Railkar, Reema; Dighe, Rajan R

2011-11-01

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions. Copyright © 2011 Wiley-Liss, Inc.

Combined effects of ankylosing spondylitis-associated ERAP1 polymorphisms outside the catalytic and peptide-binding sites on the processing of natural HLA-B27 ligands.

PubMed

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A

2014-02-14

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.

Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands*

PubMed Central

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A.

2014-01-01

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis. PMID:24352655

Characterization of recombinant yellow fever-dengue vaccine viruses with human monoclonal antibodies targeting key conformational epitopes.

PubMed

Lecouturier, Valerie; Berry, Catherine; Saulnier, Aure; Naville, Sophie; Manin, Catherine; Girerd-Chambaz, Yves; Crowe, James E; Jackson, Nicholas; Guy, Bruno

2018-04-26

The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these laboratory markers relate to vaccine efficacy and safety. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Reversal of temperature-induced conformational changes in the amyloid-beta peptide, Aβ40, by the β-sheet breaker peptides 16–23 and 17–24

PubMed Central

Hatip, Funda F Bölükbaşı; Suenaga, Midori; Yamada, Tatsuo; Matsunaga, Yoichi

2009-01-01

Background and purpose: Aggregates of the protein amyloid-beta (Aβ) play a crucial role in the pathogenesis of Alzheimer's disease (AD). Most therapeutic approaches to AD do not target Aβ, so determination of the factor(s) that facilitate aggregation and discovering agents that prevent aggregation have great potential therapeutic value. Experimental approach: We investigated ex vivo the temperature-sensitive regions of Aβ1–40 (Aβ40) and their interactions with octapeptides derived from sequences within Aβ40 –β-sheet breaker peptides (βSBP) – using enzyme-linked immunosorbent assay, and dot blot and far-UV circular dichroism (CD) spectroscopy. We measured changes within the physiological limits of temperature, using antibodies targeting epitopes 1–7, 5–10, 9–14 and 17–21 within Aβ40. Key results: Temperature-dependent conformational changes were observed in Aβ40 at epitopes 9–14 and 17–21 at 36–38 and 36–40°C respectively. The βSBPs 16–23 and 17–24, but not 15–22 and 18–25, could inhibit the changes. Moreover, βSBPs 16–23 and 17–24 increased digestion of Aβ40 by protease K, indicating a decreased aggregation of Aβ40, whereas βSBPs 15–22 and 18–25 did not increase this digestion. CD spectra revealed that β-sheet formation in Aβ40 at 38°C was reduced with βSBPs 16–23 and 17–24. Conclusions and implications: The epitopes 9–14 and 17–21 are the temperature-sensitive regions within Aβ40. The βSBPs, Aβ16–23 and 17–24 reversed temperature-induced β-sheet formation, and decreased Aβ40 aggregation. The results suggest that the 17–23 epitope of Aβ40 is crucially involved in preventing Aβ40 aggregation and consequent deposition of Aβ40 in AD brain. PMID:19785651

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

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

Do Kwon, Young; Pancera, Marie; Acharya, Priyamvada

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer boundmore » by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.« less

Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

DOE PAGES

Do Kwon, Young; Pancera, Marie; Acharya, Priyamvada; ...

2015-06-22

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. In this paper, we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer boundmore » by a single CD4 without the typical antigenic hallmarks of CD4 induction. Finally, antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.« less

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

PubMed

McCutcheon, Krista M; Gray, Julia; Chen, Natalie Y; Liu, Keyi; Park, Minha; Ellsworth, Stote; Tripp, Ralph A; Tompkins, S Mark; Johnson, Scott K; Samet, Shelly; Pereira, Lenore; Kauvar, Lawrence M

2014-01-01

Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Association study of ghrelin receptor gene polymorphisms in rheumatoid arthritis.

PubMed

Robledo, G; Rueda, B; Gonzalez-Gay, M A; Fernández, B; Lamas, J R; Balsa, A; Pascual-Salcedo, D; García, A; Raya, E; Martín, J

2010-01-01

Ghrelin is a newly characterised growth hormone (GH) releasing peptide widely distributed that may play an important role in the regulation of metabolic balance in inflammatory diseases such as rheumatoid arthritis (RA) by decreasing the pro-inflammatory Th1 responses. In this study we investigated the possible contribution of several polymorphisms in the functional Ghrelin receptor to RA susceptibility. A screening of 3 single nucleotide polymorphisms (SNPs) was performed in a total of 950 RA patients and 990 healthy controls of Spanish Caucasian origin. Genotyping of all 3 SNPs was performed by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. We observed no statistically significant deviation between RA patients and controls for the GHSR SNPs analysed. In addition, we performed a haplotype analysis that did not reveal an association with RA susceptibility. The stratification analysis for the presence of shared epitope (SE), rheumatoid factor (RF) or antibodies anti cyclic citrullinated peptide (anti-CCP) did not detect significant association of the GHSR polymorphisms with RA. These findings suggest that the GHSR gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.

Characterizing the Conformational Landscape of Flavivirus Fusion Peptides via Simulation and Experiment

PubMed Central

Marzinek, Jan K.; Lakshminarayanan, Rajamani; Goh, Eunice; Huber, Roland G.; Panzade, Sadhana; Verma, Chandra; Bond, Peter J.

2016-01-01

Conformational changes in the envelope proteins of flaviviruses help to expose the highly conserved fusion peptide (FP), a region which is critical to membrane fusion and host cell infection, and which represents a significant target for antiviral drugs and antibodies. In principle, extended timescale atomic-resolution simulations may be used to characterize the dynamics of such peptides. However, the resultant accuracy is critically dependent upon both the underlying force field and sufficient conformational sampling. In the present study, we report a comprehensive comparison of three simulation methods and four force fields comprising a total of more than 40 μs of sampling. Additionally, we describe the conformational landscape of the FP fold across all flavivirus family members. All investigated methods sampled conformations close to available X-ray structures, but exhibited differently populated ensembles. The best force field / sampling combination was sufficiently accurate to predict that the solvated peptide fold is less ordered than in the crystallographic state, which was subsequently confirmed via circular dichroism and spectrofluorometric measurements. Finally, the conformational landscape of a mutant incapable of membrane fusion was significantly shallower than wild-type variants, suggesting that dynamics should be considered when therapeutically targeting FP epitopes. PMID:26785994

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility.

PubMed

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-03-01

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein

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

Su, Hua-Poo; Golden, Joseph W.; Gittis, Apostolos G.

2007-11-25

Medical countermeasures to prevent or treat smallpox are needed due to the potential use of poxviruses as biological weapons. Safety concerns with the currently available smallpox vaccine indicate a need for research on alternative poxvirus vaccine strategies. Molecular vaccines involving the use of proteins and/or genes and recombinant antibodies are among the strategies under current investigation. The poxvirus L1 protein, encoded by the L1R open reading frame, is the target of neutralizing antibodies and has been successfully used as a component of both protein subunit and DNA vaccines. L1-specific monoclonal antibodies (e.g., mouse monoclonal antibody mAb-7D11, mAb-10F5) with potent neutralizingmore » activity bind L1 in a conformation-specific manner. This suggests that proper folding of the L1 protein used in molecular vaccines will affect the production of neutralizing antibodies and protection. Here, we co-crystallized the Fab fragment of mAb-7D11 with the L1 protein. The crystal structure of the complex between Fab-7D11 and L1 reveals the basis for the conformation-specific binding as recognition of a discontinuous epitope containing two loops that are held together by a disulfide bond. The structure of this important conformational epitope of L1 will contribute to the development of molecular poxvirus vaccines and also provides a novel target for anti-poxvirus drugs. In addition, the sequence and structure of Fab-7D11 will contribute to the development of L1-targeted immunotherapeutics.« less

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

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

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to exploremore » the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.« less

How social learning adds up to a culture: from birdsong to human public opinion

PubMed Central

Feher, Olga; Fimiarz, Daniel; Conley, Dalton

2017-01-01

ABSTRACT Distributed social learning may occur at many temporal and spatial scales, but it rarely adds up to a stable culture. Cultures vary in stability and diversity (polymorphism), ranging from chaotic or drifting cultures, through cumulative polymorphic cultures, to stable monolithic cultures with high conformity levels. What features can sustain polymorphism, preventing cultures from collapsing into either chaotic or highly conforming states? We investigate this question by integrating studies across two quite separate disciplines: the emergence of song cultures in birds, and the spread of public opinion and social conventions in humans. In songbirds, the learning process has been studied in great detail, while in human studies the structure of social networks has been experimentally manipulated on large scales. In both cases, the manner in which communication signals are compressed and filtered – either during learning or while traveling through the social network – can affect culture polymorphism and stability. We suggest a simple mechanism of a shifting balance between converging and diverging social forces to explain these effects. Understanding social forces that shape cultural evolution might be useful for designing agile communication systems, which are stable and polymorphic enough to promote gradual changes in institutional behavior. PMID:28057835

Allelic polymorphism in the T cell receptor and its impact on immune responses.

PubMed

Gras, Stephanie; Chen, Zhenjun; Miles, John J; Liu, Yu Chih; Bell, Melissa J; Sullivan, Lucy C; Kjer-Nielsen, Lars; Brennan, Rebekah M; Burrows, Jacqueline M; Neller, Michelle A; Khanna, Rajiv; Purcell, Anthony W; Brooks, Andrew G; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R

2010-07-05

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

PubMed Central

Korkut, Anil; Hendrickson, Wayne A.

2012-01-01

HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes. PMID:23300605

Virus-mimetic nanovesicles as a versatile antigen-delivery system

PubMed Central

Zhang, Pengfei; Chen, Yixin; Zeng, Yun; Shen, Chenguang; Li, Rui; Guo, Zhide; Li, Shaowei; Zheng, Qingbing; Chu, Chengchao; Wang, Zhantong; Zheng, Zizheng; Tian, Rui; Ge, Shengxiang; Zhang, Xianzhong; Xia, Ning-Shao; Liu, Gang; Chen, Xiaoyuan

2015-01-01

It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses. PMID:26504197

Structure-Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

PubMed Central

Pelay-Gimeno, Marta; Glas, Adrian; Koch, Oliver; Grossmann, Tom N

2015-01-01

Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure-based design of PPI inhibitors through stabilizing or mimicking turns, β-sheets, and helices. PMID:26119925

Methods for detection of ataxia telangiectasia mutations

DOEpatents

Gatti, Richard A.

2005-10-04

The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

Modified SSCP method using sequential electrophoresis of multiple nucleic acid segments

DOEpatents

Gatti, Richard A.

2002-10-01

The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

[Research progress of molecular genetic analysis in Schistosoma variation].

PubMed

Zheng, Su-Yue; Li, Fei

2014-02-01

The development of molecular biology techniques makes important contributions to the researches of heritable variation of Schistosoma. In recent years, the molecular genetic analysis in the Schistosoma variation researches mainly includes the restriction fragment length polymorphism (RFLP), random amplified polymorphism technology (RAPD), microsatellite anchored PCR (SSR-PCR), and polymerase reaction single-strand conformation polymorphism (PCR-SSCP). This article reviews the research progress of molecular genetic analysis in Schistosoma variation in recent years.

Genetic and immunological comparison of the cladoceran parasite Pasteuria ramosa with the nematode parasite Pasteuria penetrans.

PubMed

Schmidt, Liesbeth M; Mouton, Laurence; Nong, Guang; Ebert, Dieter; Preston, James F

2008-01-01

Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans▿

PubMed Central

Schmidt, Liesbeth M.; Mouton, Laurence; Nong, Guang; Ebert, Dieter; Preston, James F.

2008-01-01

Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts. PMID:17933927

Ocular findings associated with a Cys39Arg mutation in the Norrie disease gene.

PubMed

Joos, K M; Kimura, A E; Vandenburgh, K; Bartley, J A; Stone, E M

1994-12-01

To diagnose the carriers and noncarriers in a family affected with Norrie disease based on molecular analysis. Family members from three generations, including one affected patient, two obligate carriers, one carrier identified with linkage analysis, one noncarrier identified with linkage analysis, and one female family member with indeterminate carrier status, were examined clinically and electrophysiologically. Linkage analysis had previously failed to determine the carrier status of one female family member in the third generation. Blood samples were screened for mutations in the Norrie disease gene with single-strand conformation polymorphism analysis. The mutation was characterized by dideoxy-termination sequencing. Ophthalmoscopy and electroretinographic examination failed to detect the carrier state. The affected individuals and carriers in this family were found to have a transition from thymidine to cytosine in the first nucleotide of codon 39 of the Norrie disease gene, causing a cysteine-to-arginine mutation. Single-strand conformation polymorphism analysis identified a patient of indeterminate status (by linkage) to be a noncarrier of Norrie disease. Ophthalmoscopy and electroretinography could not identify carriers of this Norrie disease mutation. Single-strand conformation polymorphism analysis was more sensitive and specific than linkage analysis in identifying carriers in this family.

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.

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.

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.

Pharmaceutical polymorph control in a drug-mimetic supramolecular gel† †Electronic supplementary information (ESI) available: Synthetic and crystallographic experimental details, rheology, full crystallization and calculation details. See DOI: 10.1039/c6sc04126d Click here for additional data file.

PubMed Central

Foster, Jonathan A.; Damodaran, Krishna K.; Maurin, Antoine; Thompson, Hugh P. G.; Cameron, Gary J.; Bernal, Jenifer Cuesta

2017-01-01

We report the synthesis of a bis(urea) gelator designed to specifically mimic the chemical structure of the highly polymorphic drug substance ROY. Crystallization of ROY from toluene gels of this gelator results in the formation of the metastable red form instead of the thermodynamic yellow polymorph. In contrast, all other gels and solution control experiments give the yellow form. Conformational and crystal structure prediction methods have been used to propose the structure of the gel and show that the templation of the red form by the targeted gel results from conformational matching of the gelator to the ROY substrate coupled with overgrowth of ROY onto the local periodic structure of the gel fibres. PMID:28451150

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers

PubMed Central

Morris, Charles D.; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J.; Tang, Jeffrey; Sok, Devin; Burton, Dennis R.; Law, Mansun; Ward, Andrew B.

2017-01-01

ABSTRACT Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. PMID:28246356

An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene

PubMed Central

Sun, Zhiwu; Zhu, Yun; Wang, Qian; Ye, Ling; Dai, Yanyan; Su, Shan; Yu, Fei; Ying, Tianlei; Yang, Chinglai; Jiang, Shibo; Lu, Lu

2016-01-01

After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design. PMID:27329850

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

Relating conformation to function in integrin α5β1.

PubMed

Su, Yang; Xia, Wei; Li, Jing; Walz, Thomas; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

2016-07-05

Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

Conformational and Thermal Stability Improvements for the Large-Scale Production of Yeast-Derived Rabbit Hemorrhagic Disease Virus-Like Particles as Multipurpose Vaccine

PubMed Central

Méndez, Lídice; González, Nemecio; Parra, Francisco; Martín-Alonso, José M.; Limonta, Miladys; Sánchez, Kosara; Cabrales, Ania; Estrada, Mario P.; Rodríguez-Mallón, Alina; Farnós, Omar

2013-01-01

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans. PMID:23460801

A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

PubMed Central

Lee, Hyunwook; Brendle, Sarah A.; Bywaters, Stephanie M.; Guan, Jian; Ashley, Robert E.; Yoder, Joshua D.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.

2014-01-01

ABSTRACT Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used by H16.V5. PMID:25392224

Structural analysis of linear and conformational epitopes of allergens

PubMed Central

Ivanciuc, Ovidiu; Schein, Catherine H.; Garcia, Tzintzuni; Oezguen, Numan; Negi, Surendra S.; Braun, Werner

2009-01-01

In many countries regulatory agencies have adopted safety guidelines, based on bioinformatics rules from the WHO/FAO and EFSA recommendations, to prevent potentially allergenic novel foods or agricultural products from reaching consumers. We created the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to combine data that had previously been available only as flat files on Web pages or in the literature. SDAP was designed to be user friendly, to be of maximum use to regulatory agencies, clinicians, as well as to scientists interested in assessing the potential allergenic risk of a protein. We developed methods, unique to SDAP, to compare the physicochemical properties of discrete areas of allergenic proteins to known IgE epitopes. We developed a new similarity measure, the property distance (PD) value that can be used to detect related segments in allergens with clinical observed crossreactivity. We have now expanded this work to obtain experimental validation of the PD index as a quantitative predictor of IgE cross-reactivity, by designing peptide variants with predetermined PD scores relative to known IgE epitopes. In complementary work we show how sequence motifs characteristic of allergenic proteins in protein families can be used as fingerprints for allergenicity. PMID:19121639

Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

DOE PAGES

Pan, Ruimin; Chen, Yuxin; Vaine, Michael; ...

2015-07-15

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitopemore » peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI 439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.« less

The antigen-binding site of an N-propionylated polysialic acid-specific antibody protective against group B meningococci is consistent with extended epitopes.

PubMed

Johal, Asha R; Jarrell, Harold C; Letts, James A; Khieu, Nam Huan; Landry, Roxanne C; Jachymek, Wojciech; Yang, Qingling; Jennings, Harold J; Brisson, Jean-Robert; Evans, Stephen V

2013-08-01

Monoclonal antibodies 13D9 and 6B9 are both specific for N-propionylated polysialic acid (NPrPSA); however, while 13D9 is protective against meningitis caused by group B meningococci and Escherichia coli capsular type K1 infection, 6B9 is not. The crystal structures of the Fabs from the two antibodies determined at 2.06 and 2.45 Å resolutions, respectively, reveal markedly different combining sites, where only the surface of 13D9 is consistent with the recognition of extended helical epitopes known to exist in the capsular polysaccharides of etiological agents of meningitis. Interestingly, complementarity determining region H2 on 13D9 lies in a non-canonical conformation that docking studies show is a critical feature in the generation of negative free energy of binding. Finally, the model of extended NPrPSA decasaccharide bound to 13D9 derived from docking studies is consistent with saturation transfer difference nuclear magnetic resonance experiments. Together, these results provide further evidence that extended epitopes have the ability to break immune tolerance associated with the polysialic acid capsule of these pathogens.

Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

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

Pan, Ruimin; Chen, Yuxin; Vaine, Michael

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitopemore » peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI 439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.« less

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

PubMed

Morris, Charles D; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J; Tang, Jeffrey; Sok, Devin; Burton, Dennis R; Law, Mansun; Ward, Andrew B; He, Linling; Zhu, Jiang

2017-02-28

Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. IMPORTANCE Both epitope-focused and trimer-based strategies are currently being explored in HIV-1 vaccine development, which aims to elicit broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the viral envelope (Env). However, little is known about the differences in antibody response to these bNAb targets presented by foreign scaffolds and native Env. In this study, a systematic effort was undertaken to design multivalent epitope scaffolds and soluble gp140.681 trimers with a complete antigenic surface, and to comparatively analyze the antibody responses elicited by these antigens to the N332 supersite and MPER in a mouse model. This study will inform both epitope-focused and trimer-based vaccine design and will facilitate integration of the two vaccine strategies. Copyright © 2017 Morris et al.

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

A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury.

PubMed

Thiele, Jan R; Zeller, Johannes; Kiefer, Jurij; Braig, David; Kreuzaler, Sheena; Lenz, Yvonne; Potempa, Lawrence A; Grahammer, Florian; Huber, Tobias B; Huber-Lang, M; Bannasch, Holger; Stark, G Björn; Peter, Karlheinz; Eisenhardt, Steffen U

2018-01-01

C-reactive protein circulates as a pentameric protein (pCRP). pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP)]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI). Rat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN) was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine)-hexane (1,6-bisPC), which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS) induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68 + leukocytes. The application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP). Structurally altered CRP induces leukocyte-endothelial interaction and induces ROS formation in leukocytes, the latter can be abrogated by blocking lipid raft-dependent signaling pathways with Nystatin. Stabilizing pCRP in its native pentameric state abrogates these pro-inflammatory effects. Importantly, these findings are confirmed in human IRI challenged muscle tissue. These results suggest that CRP is a potent modulator of IRI. Stabilizing the native pCRP conformation represents a promising anti-inflammatory therapeutic strategy by attenuation of leukocyte recruitment and ROS formation, the primary pathomechanisms of IRI.

Heterogeneity in the A33 Protein Impacts the Cross-Protective Efficacy of a Candidate Smallpox DNA Vaccine

DTIC Science & Technology

2008-01-01

include myocarditis, progressive vaccinia, eczema vaccinatum, and even death (Bray, 2003; Cassimatis et al., 2004; Eckart et al., 2004;Kretzschmaret...hundreds of genes, many of which encode for immunomodulatory molecules or molecules with unknown function . The safety risks posed by these molecules...1G10 or function indirectly by impacting the conformation of the physical epitope. However, findings in the literature support the conclusion that

Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia

PubMed Central

Hernández-Martínez, Miguel Ángel; Escalante, Ananías A.; Arévalo-Herrera, Myriam; Herrera, Sócrates

2011-01-01

Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly → Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4+, and T-CD8+ cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions. PMID:21292878

Mapping epitopes and antigenicity by site-directed masking

NASA Astrophysics Data System (ADS)

Paus, Didrik; Winter, Greg

2006-06-01

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

PubMed

Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

2014-02-01

Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine. Copyright © 2013 Elsevier GmbH. All rights reserved.

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.

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure*

PubMed Central

Dixon, Ann M.; Drake, Lisa; Hughes, Kelly T.; Sargent, Elizabeth; Hunt, Danielle; Harton, Jonathan A.; Drake, James R.

2014-01-01

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2+ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2+ I-Ak conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2+ versus Ia.2− I-Ak class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response. PMID:24619409

How social learning adds up to a culture: from birdsong to human public opinion.

PubMed

Tchernichovski, Ofer; Feher, Olga; Fimiarz, Daniel; Conley, Dalton

2017-01-01

Distributed social learning may occur at many temporal and spatial scales, but it rarely adds up to a stable culture. Cultures vary in stability and diversity (polymorphism), ranging from chaotic or drifting cultures, through cumulative polymorphic cultures, to stable monolithic cultures with high conformity levels. What features can sustain polymorphism, preventing cultures from collapsing into either chaotic or highly conforming states? We investigate this question by integrating studies across two quite separate disciplines: the emergence of song cultures in birds, and the spread of public opinion and social conventions in humans. In songbirds, the learning process has been studied in great detail, while in human studies the structure of social networks has been experimentally manipulated on large scales. In both cases, the manner in which communication signals are compressed and filtered - either during learning or while traveling through the social network - can affect culture polymorphism and stability. We suggest a simple mechanism of a shifting balance between converging and diverging social forces to explain these effects. Understanding social forces that shape cultural evolution might be useful for designing agile communication systems, which are stable and polymorphic enough to promote gradual changes in institutional behavior. © 2017. Published by The Company of Biologists Ltd.

Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule

PubMed Central

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M.; Malmsten, Martin; Mörgelin, Matthias

2013-01-01

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity. PMID:23656734

Proteolytic activation transforms heparin cofactor II into a host defense molecule.

PubMed

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

2013-06-15

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

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

Balance between transmitted HLA preadapted and nonassociated polymorphisms is a major determinant of HIV-1 disease progression.

PubMed

Mónaco, Daniela C; Dilernia, Dario A; Fiore-Gartland, Andrew; Yu, Tianwei; Prince, Jessica L; Dennis, Kristine K; Qin, Kai; Schaefer, Malinda; Claiborne, Daniel T; Kilembe, William; Tang, Jianming; Price, Matt A; Farmer, Paul; Gilmour, Jill; Bansal, Anju; Allen, Susan; Goepfert, Paul; Hunter, Eric

2016-09-19

HIV-1 adapts to a new host through mutations that facilitate immune escape. Here, we evaluate the impact on viral control and disease progression of transmitted polymorphisms that were either preadapted to or nonassociated with the new host's HLA. In a cohort of 169 Zambian heterosexual transmission pairs, we found that almost one-third of possible HLA-linked target sites in the transmitted virus Gag protein are already adapted, and that this transmitted preadaptation significantly reduced early immune recognition of epitopes. Transmitted preadapted and nonassociated polymorphisms showed opposing effects on set-point VL and the balance between the two was significantly associated with higher set-point VLs in a multivariable model including other risk factors. Transmitted preadaptation was also significantly associated with faster CD4 decline (<350 cells/µl) and this association was stronger after accounting for nonassociated polymorphisms, which were linked with slower CD4 decline. Overall, the relative ratio of the two classes of polymorphisms was found to be the major determinant of CD4 decline in a multivariable model including other risk factors. This study reveals that, even before an immune response is mounted in the new host, the balance of these opposing factors can significantly influence the outcome of HIV-1 infection. © 2016 Mónaco et al.

Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance.

PubMed

Bailey, Justin R; Wasilewski, Lisa N; Snider, Anna E; El-Diwany, Ramy; Osburn, William O; Keck, Zhenyong; Foung, Steven K H; Ray, Stuart C

2015-01-01

For hepatitis C virus (HCV) and other highly variable viruses, broadly neutralizing mAbs are an important guide for vaccine development. The development of resistance to anti-HCV mAbs is poorly understood, in part due to a lack of neutralization testing against diverse, representative panels of HCV variants. Here, we developed a neutralization panel expressing diverse, naturally occurring HCV envelopes (E1E2s) and used this panel to characterize neutralizing breadth and resistance mechanisms of 18 previously described broadly neutralizing anti-HCV human mAbs. The observed mAb resistance could not be attributed to polymorphisms in E1E2 at known mAb-binding residues. Additionally, hierarchical clustering analysis of neutralization resistance patterns revealed relationships between mAbs that were not predicted by prior epitope mapping, identifying 3 distinct neutralization clusters. Using this clustering analysis and envelope sequence data, we identified polymorphisms in E2 that confer resistance to multiple broadly neutralizing mAbs. These polymorphisms, which are not at mAb contact residues, also conferred resistance to neutralization by plasma from HCV-infected subjects. Together, our method of neutralization clustering with sequence analysis reveals that polymorphisms at noncontact residues may be a major immune evasion mechanism for HCV, facilitating viral persistence and presenting a challenge for HCV vaccine development.

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility

PubMed Central

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-01-01

Summary The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcβ1Π4(Neu5Acα2Π3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to β-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcβ1Π4Gal linkage in GM2 was converted to the GalNAcβ1Π6Gal, both the GalNAc and the Neu5Ac became susceptible to β-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates. PMID:17967427

Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations.

PubMed

Szczepina, Monica G; Bleile, Dustin W; Pinto, B Mario

2011-10-04

Saturation transfer difference (STD)-NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O-polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD™ Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA-ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD-NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor-ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R(eff), was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody-bound solution conformation of the peptide which also differed from the bound conformation determined by X-ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD-NMR intensity-restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O-polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build-up, which, in turn, hindered a rigorous analysis via full STD build-up curves. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solid-state NMR and IR for the analysis of pharmaceutical solids: polymorphs of fosinopril sodium.

PubMed

Brittain, H G; Morris, K R; Bugay, D E; Thakur, A B; Serajuddin, A T

1993-01-01

The two polymorphic modifications of fosinopril sodium have been characterized as to their differences in melting behaviour, powder X-ray diffraction patterns, Fourier transform infrared spectra (FTIR), and solid-state 31P- and 13C-NMR spectra. The polymorphs were found to be enantiotropically related based upon melting point, heat of fusion, and solution mediated transformation data. Analysis of the solid-state FTIR and 13C-NMR data indicated that the environment of the acetal side chain of fosinopril sodium differed in two polymorphs, and that there might be cis-trans isomerization about the C6-N peptide bond. These conformational differences are postulated as the origin of the observed polymorphism.

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

PubMed Central

Ader, Nadine; Brindley, Melinda; Avila, Mislay; Örvell, Claes; Horvat, Branka; Hiltensperger, Georg; Schneider-Schaulies, Jürgen; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.

2013-01-01

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering. PMID:23077316

Quarterly Performance/Technical Report of the National Marrow Donor Program

DTIC Science & Technology

2009-02-10

and epitope level matching for HLA-DPB1. Submitted. o Z Shamim , L Ryder, M Haagenson , S Spellman, T Wang, S Lee, K Müller. Polymorphism in the...3, 4 6. AUTHOR( S ) Setterholm, Michelle 5f. WORK UNIT NUMBER N/A 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES) National Marrow Donor...Program 3001 Broadway St., N.E., Ste. 500 Minneapolis, MN 55413 8. PERFORMING ORGANIZATION REPORT NUMBER N/A 10. SPONSOR/MONITOR’S ACRONYM( S ) ONR 9

A role for extracellular amastigotes in the immunopathology of Chagas disease.

PubMed

Scharfstein, J; Morrot, A

1999-01-01

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

Polymorphs and solvatomorphs of azilsartan medoxomil: Elucidation of solvent-induced construction and conformational diversity

NASA Astrophysics Data System (ADS)

Zhang, Xian-Rui; He, Sai-Fei; Zhang, Shuo; Li, Jing; Li, Shan; Liu, Jin-Song; Zhang, Lei

2017-02-01

Two polymorphs (AM-A and AM-B) of azilsartan medoxomil (AM) and four AM solvatomorphs with toluene (AM-TOL), 1,4-dioxane (AM-DIO), chloroform (AM-TCM) and N,N-dimethylacetamide (AM-DMA) have been prepared by the hydrolysis of azilsartan medoxomil potassium in aqueous-organic solutions. In the crystal structures of two polymorphs and three solvatomorphs (AM-TOL, AM-DIO and AM-TCM), two asymmetric AM molecules form the dimeric cycle-like structures via intermolecular Nsbnd H⋯N hydrogen bonds in R22 (26) ring, while AM-DMA shows intramolecular Nsbnd H⋯O hydrogen bond between AM and DMA molecules. The hydrogen bonds (Csbnd H⋯O or Csbnd H⋯N) and π···π (or Csbnd H···π) interactions are helpful to stabilize the conformational diversity of AM. The solvent-induced experiment shows that solvent molecules have great influence on the solvatomorph formation and DIO can form the most steady solvatomorph than other solvents. The thermal study demonstrates that toluene molecules in three solvatomorphs (AM-TOL, AM-DIO and AM-TCM) are the most difficult to remove from the cage. Our results illustrate that the solvent plays significant role in tuning the size of the cage and producing the conformational diversity of AM molecules.

Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

PubMed Central

Provenzano, Maurizio; Mocellin, Simone; Bonginelli, Paola; Nagorsen, Dirk; Kwon, Seog-Woon; Stroncek, David

2003-01-01

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization. PMID:14675481

Changes in structural and antigenic properties of proteins by radiation

NASA Astrophysics Data System (ADS)

Kume, Tamikazu; Matsuda, Tsukasa

1995-08-01

Radiation effect on structural and antigenic properties of proteins (0.2% in 0.01 M phosphate buffer, pH 7.4) were investigated using ovalbumin (OVA) and bovine serum albumin (BSA). Aggregation of OVA and BSA was induced by radiation and the molecular mass increased significantly in N 2. Significant changes in surface hydrophobicity and [ θ] 222 nm of CD were also observed by radiation showing the destruction of secondary structure of proteins. Antigenicity of irradiated OVA measured by the method of immunodiffusion was decreased by radiation, and the reactivity to anti-OVA antibody was almost diminished at 8 kGy in N 2 and 4 kGy in O 2, respectively. The reactivity of BSA was diminished at 4 kGy both in N 2 and O 2. Changes in hydrophobicity of OVA did not correspond to the decrease in antigenicity, whereas the changes in [ θ] 222 nm relatively well corresponded to the antigenicity. The SDS-PAGE and immunoblotting analysis showed that radiation at higher doses induced the production of protein aggregates and degraded fragments with reactivity to the specific antibodies. These results suggest that the main part of conformation-dependent antigenic structure (conformational epitope) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes), remains even at higher dose.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

PubMed Central

Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

2014-01-01

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

Are linear AChR epitopes the real culprit in ocular myasthenia gravis?

PubMed

Wu, Xiaorong; Tüzün, Erdem

2017-02-01

Extraocular muscle weakness occurs in most of the myasthenia gravis (MG) patients and it is often the initial complaint. Approximately 10-20% of MG patients with extraocular muscle weakness display only ocular symptoms and rest of the patients subsequently develop generalized muscle weakness. It is not entirely clear why some MG patients develop only ocular symptoms and why extraocular muscle weakness almost always precedes generalized muscle weakness. These facts are often explained by increased susceptibility of extraocular muscles due to their reduced endplate safety factor and lower complement inhibitor expression. Findings of a recently developed animal model of ocular MG suggest that additional factors might be in play. While immunization of HLA transgenic and wild-type (WT) mice with the native acetylcholine receptor (AChR) pentamer carrying conformational epitopes generates severe generalized muscle weakness, immunization of the same mouse strains with recombinant unfolded AChR subunits containing linear epitopes induces ptosis with or without mild generalized muscle weakness. Notably, immunization of mice with deficient T helper cell-mediated antigen presentation with recombinant AChR subunits or whole native AChR pentamer also induces ocular symptoms, AChR-reactive B cells and AChR antibodies. Based on these findings, we hypothesize that ocular symptoms observed in the earlier stages of MG might be triggered by linear and non-conformational AChR epitopes expressed by thymic cells or invading microorganisms. This initial AChR autoimmunity might be managed by T cell-independent and B cell mediated mechanisms yielding low affinity AChR antibodies. These antibodies are putatively capable of inducing muscle weakness only in extraocular muscles which have increased vulnerability due to their inherent biological properties. After this initial attack, as AChR bearing immune complexes form and the immune system gains access to the native AChR expressed by muscle and thymic myoid cells, a more robust anti-AChR autoimmunity develops giving way to high affinity AChR antibodies, thymic germinal center formation and severe generalized muscle weakness. Accurate characterization of chain if events leading to ocular and generalized symptoms in MG might enable development of novel therapeutics that might prevent the transition from mild ocular symptoms to severe generalized weakness in earlier stages of the disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of structural variations in the carboxyl terminus of Alzheimer's disease-associated βA4[1–42] amyloid using a monoclonal antibody

PubMed Central

Jayasena, U L H R; Gribble, S K; Mckenzie, A; Beyreuther, K; Masters, C L; Underwood, J R

2001-01-01

The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the βA4 (Aß) polypeptide represents a major component with the C-terminal 39–43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma βA4[35–43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues βA438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human βA4[1–42]. Although the βA438[GVV]40 sequence is present within the C-termini of human βA4[1–40] and βA4[1–43] and the βA4-containing region of human APP, the βA4[35–43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the βA438[GVV]40 epitope as presented within the βA4[1–42] sequence. The βA4[1–42] epitope bound by MoAb 3B9 is sensitive to heating (100°C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of βA4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of βA4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique βA4[1–42] conformation in the development of Alzheimer's disease. PMID:11422208

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

Detection of AGXT bgene mutations by denaturing high-performance liquid chromatography for diagnosis of hyperoxaluria type 1.

PubMed

Pirulli, D; Giordano, M; Lessi, M; Spanò, A; Puzzer, D; Zezlina, S; Boniotto, M; Crovella, S; Florian, F; Marangella, M; Momigliano-Richiardi, P; Savoldi, S; Amoroso, A

2001-06-01

Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT. The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type I previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.

Structural study of piracetam polymorphs and cocrystals: crystallography redetermination and quantum mechanics calculations.

PubMed

Tilborg, Anaëlle; Jacquemin, Denis; Norberg, Bernadette; Perpète, Eric; Michaux, Catherine; Wouters, Johan

2011-12-01

Pharmaceutical compounds are mostly developed as solid dosage forms containing a single-crystal form. It means that the selection of a particular crystal state for a given molecule is an important step for further clinical outlooks. In this context, piracetam, a pharmaceutical molecule known since the sixties for its nootropic properties, is considered in the present work. This molecule is analyzed using several experimental and theoretical approaches. First, the conformational space of the molecule has been systematically explored by performing a quantum mechanics scan of the two most relevant dihedral angles of the lateral chain. The predicted stable conformations have been compared to all the reported experimental geometries retrieved from the Cambridge Structural Database (CSD) covering polymorphs and cocrystals structures. In parallel, different batches of powders have been recrystallized. Under specific conditions, single crystals of polymorph (III) of piracetam have been obtained, an outcome confirmed by crystallographic analysis. © 2011 International Union of Crystallography. Printed in Singapore – all rights reserved.

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.

Development of an ELA-DRA gene typing method based on pyrosequencing technology.

PubMed

Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G

2008-11-01

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

Self-assembled monolayers of shape-persistent macrocycles on graphite: interior design and conformational polymorphism.

PubMed

Vollmeyer, Joscha; Eberhagen, Friederike; Höger, Sigurd; Jester, Stefan-S

2014-01-01

Three shape-persistent naphthylene-phenylene-acetylene macrocycles of identical backbone structures and extraannular substitution patterns but different (empty, apolar, polar) nanopore fillings are self-assembled at the solid/liquid interface of highly oriented pyrolytic graphite and 1,2,4-trichlorobenzene. Submolecularly resolved images of the resulting two-dimensional (2D) crystalline monolayer patterns are obtained by in situ scanning tunneling microscopy. A concentration-dependent conformational polymorphism is found, and open and more dense packing motifs are observed. For all three compounds alike lattice parameters are found, therefore the intermolecular macrocycle distances are mainly determined by their size and symmetry. This is an excellent example that the graphite acts as a template for the macrocycle organization independent from their specific interior.

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

PubMed

Williams, D J; Newson, J; Naessens, J

1990-03-01

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.

Venom allergen-like protein 28 in Clonorchis sinensis: four epitopes on its surface and the potential role of Cys124 for its conformational stability.

PubMed

Lee, Myoung-Ro; Yoo, Won Gi; Kim, Yu Jung; Chung, Eun Ju; Cho, Shin-Hyeong; Ju, Jung-Won

2018-06-06

Venom allergen-like (VAL) proteins are important to host-parasite interactions. We previously demonstrated that a Clonorchis sinensis VAL (CsVAL) protein-derived synthetic peptide suppresses allergic and inflammatory responses. However, little is known regarding the physicochemical and antigenic properties of CsVAL proteins. Here, we identified a novel 194 amino acid VAL protein, named C. sinensis VAL 28 (CsVAL28), and characterized its functional motifs and structural details as a new member of the CAP superfamily. Unlike members of the Schistosoma mansoni VAL (SmVAL) family, CsVAL28 has a single CAP1 motif and six highly conserved disulfide bond-forming cysteines. Tertiary models of wild-type CsVAL28 and mutants were built using SmVAL4 as template via homology modeling. Normal mode analysis predicted that disulfide bond breaking by mutation of cysteine 124 to serine would greatly affect protein mobility. Four major immunoreactive linear epitopes were identified in the surface-exposed region or its vicinity via epitope mapping, using sera from clonorchiasis patients and healthy controls. Our findings provide in-depth knowledge on the structure-function properties of VAL proteins and may help determine highly antigenic regions for developing new diagnostic approaches.

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

PubMed Central

Bosco, Daryl A.; Morfini, Gerardo; Karabacak, N. Murat; Song, Yuyu; Gros-Louis, Francois; Pasinelli, Piera; Goolsby, Holly; Fontaine, Benjamin A.; Lemay, Nathan; McKenna-Yasek, Diane; Frosch, Matthew P.; Agar, Jeffery N.; Julien, Jean-Pierre; Brady, Scott T.; Brown, Robert H.

2010-01-01

Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS. PMID:20953194

HLA-F and MHC-I Open Conformers Cooperate in a MHC-I Antigen Cross-Presentation Pathway

PubMed Central

Goodridge, Jodie P.; Lee, Ni; Burian, Aura; Pyo, Chul-Woo; Tykodi, Scott S.; Warren, Edus H.; Yee, Cassian; Riddell, Stanley R.

2013-01-01

Peptides that are presented by MHC class I (MHC-I) are processed from two potential sources, as follows: newly synthesized endogenous proteins for direct presentation on the surface of most nucleated cells and exogenous proteins for cross-presentation typically by professional APCs. In this study, we present data that implicate the nonclassical HLA-F and open conformers of MHC-I expressed on activated cells in a pathway for the presentation of exogenous proteins by MHC-I. This pathway is distinguished from the conventional endogenous pathway by its independence from TAP and tapasin and its sensitivity to inhibitors of lysosomal enzymes, and further distinguished by its dependence on MHC-I allotype-specific epitope recognition for Ag uptake. Thus, our data from in vitro experiments collectively support a previously unrecognized model of Ag cross-presentation mediated by HLA-F and MHC-I open conformers on activated lymphocytes and monocytes, which may significantly contribute to the regulation of immune system functions and the immune defense. PMID:23851683

Two polymorphs of safinamide, a selective and reversible inhibitor of monoamine oxidase B.

PubMed

Ravikumar, Krishnan; Sridhar, Balasubramanian

2010-06-01

Two polymorphs of safinamide {systematic name: (2S)-2-[4-(3-fluorobenzyloxy)benzylamino]propionamide}, C(17)H(19)FN(2)O(2), a potent selective and reversible monoamine oxidase B (MAO-B) inhibitor, are described. Both forms are orthorhombic and regarded as conformational polymorphs due to the differences in the orientation of the 3-fluorobenzyloxy and propanamide groups. Both structures pack with layers in the ac plane. In polymorph (I), the layers have discrete wide and narrow regions which are complementary when located next to adjacent layers. In polymorph (II), the layer has long flanges protruding from each side, which interdigitate when packed with the adjacent layers. N-H...O hydrogen bonds are present in both structures, whereas N-H...F hydrogen bonding is seen in polymorph (I), while N-H...N hydrogen bonding is seen in polymorph (II).

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

Tempest in a sugar-coated lab vial.

PubMed

Dragun, Duska; Philippe, Aurélie

2018-06-23

Angiotensin II type 1 receptor (AT 1 R) is a classical G-protein-coupled-receptor (GPCR) displaying complex structure consisting of 7-transmembrane helices connected by intracellular and extracellular loops. Beside Angiotensin II binding within transmembrane sites and mechanically induced ligand free activation, AT 1 R can be also activated by agonistic autoantibodies (AT 1 R-Ab) recognizing conformational epitopes contained in the second extracellular loop. Direct pathophysiologic involvement of AT 1 R-Abs is well established in several autoimmune contexts and organ transplantation (1). A commercially available sandwich ELISA appreciating native receptor conformation relies on cell membrane AT 1 R extracts from human AT 1 R overexpressing Chinese hamster ovary (CHO) cells as a solid phase. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Molecular Basis of Allele-Specific Efficacy of a Blood-Stage Malaria Vaccine: Vaccine Development Implications

PubMed Central

Ouattara, Amed; Takala-Harrison, Shannon; Thera, Mahamadou A.; Coulibaly, Drissa; Niangaly, Amadou; Saye, Renion; Tolo, Youssouf; Dutta, Sheetij; Heppner, D. Gray; Soisson, Lorraine; Diggs, Carter L.; Vekemans, Johan; Cohen, Joe; Blackwelder, William C.; Dube, Tina; Laurens, Matthew B.; Doumbo, Ogobara K.; Plowe, Christopher V.

2013-01-01

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02A, a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02A had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen. PMID:23204168

Three reversible polymorphic copper(I) complexes triggered by ligand conformation: insights into polymorphic crystal habit and luminescent properties.

PubMed

Chai, Wenxiang; Hong, Mingwei; Song, Li; Jia, Guohua; Shi, Hongsheng; Guo, Jiayu; Shu, Kangying; Guo, Bing; Zhang, Yicheng; You, Wenwu; Chen, Xueyuan

2015-05-04

Three luminescent polymorphs based on a new copper(I) complex Cu(2-QBO)(PPh3)PF6 (1, PPh3 = triphenylphosphine, 2-QBO = 2-(2'-quinolyl)benzoxazole) have been synthesized and characterized by FT-IR, UV-vis, elemental analyses, and single-crystal X-ray diffraction analyses. Each polymorph can reversibly convert from one to another through appropriate procedures. Interestingly, such interconversion can be distinguished by their intrinsic crystal morphologies and colors (namely α, dark yellow plate, β, orange block, γ, light yellow needle) as well as photoluminescent (PL) properties. X-ray crystal structure analyses of these three polymorphs show three different supramolecular structures from 1D to 3D, which are expected to be responsible for the formation of three different crystal morphologies such as needle, plate, and block. Combination of the experimental data with DFT calculations on these three polymorphs reveals that the polymorphic interconversion is triggered by the conformation isomerization of the 2-QBO ligand and can be successfully controlled by the polarity of the process solvents (affecting the molecular dipole moment) and thermodynamics (affecting the molecular total energy). It is also found that the different crystal colors of polymorphs and their PL properties are derived from different θ values (dihedral angle between benzoxazolyl and quinolyl group of the 2-QBO ligand) and P-Cu-P angles based on TD-DFT calculations. Moreover, an interesting phase interconversion between γ and β has also been found under different temperature, and this result is consistent with the DFT calculations in which the total energy of β is larger than that of γ. This polymorphism provides a good model to study the relationship between the structure and the physical properties in luminescent copper(I) complexes as well as some profound insights into their PL properties.

Polymorphism in Energetic Materials

DTIC Science & Technology

2008-01-01

2Department of Chemistry, Howard University Polymorphism often occurs in energetic materials. Differences in the forms range from conformational changes in...these two areas. rayMond J. ButchEr is a professor of inorganic and structural chemistry at Howard University , Washington, DC. He has worked at Howard ... University since 1977 and has been associated with the NRL Laboratory for Structure of Matter since 1989 (primarily during the summer months as an

Conformational studies of immunodominant myelin basic protein 1-11 analogues using NMR and molecular modeling

NASA Astrophysics Data System (ADS)

Laimou, Despina; Lazoura, Eliada; Troganis, Anastassios N.; Matsoukas, Minos-Timotheos; Deraos, Spyros N.; Katsara, Maria; Matsoukas, John; Apostolopoulos, Vasso; Tselios, Theodore V.

2011-11-01

Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP1-11) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP1-11[4A]) or a tyrosine residue (Ac-MBP1-11[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-Au was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln3 residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-Au complex, has a different orientation in the mutated analogues especially in the Ac-MBP1-11[4A] peptide. In particular the side chain of Gln3 is not solvent exposed as for the native Ac-MBP1-11 and it is not available for interaction with the TCR.

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

PubMed

Pesavento, Joseph B; Crawford, Sue E; Roberts, Ed; Estes, Mary K; Prasad, B V Venkataram

2005-07-01

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Polymorphism and haplotype analyses of swine leukocyte antigen DQA exons 2, 3, 4, and their associations with piglet diarrhea in Chinese native pig.

PubMed

Huang, X Y; Yang, Q L; Yuan, J H; Gun, S B

2015-09-08

In this study, 290 Chinese native Yantai black pig piglets were investigated to identify gene polymorphisms, for haplotype reconstruction, and to determine the association between piglet diarrhea and swine leukocyte antigen (SLA) class II DQA exons 2, 3, and 4 by polymerase chain reaction-single stranded conformational polymorphism and cloning sequencing. The results showed that the 5, 8, and 7 genotypes were identified from SLA-DQA exon 2, 3, and 4, respectively, based on the single-stranded conformational polymorphism banding patterns and found a novel allele D in exon 2 and 2 novel mutational sites of allele C (c.4828T>C) and allele F (c.4617T>C) in exon 3. Polymorphism information content testing showed that exon 2 was moderately polymorphic and that exons-3 and -4 loci were highly polymorphic. The piglet diarrhea scores for genotypes AB (1.40 ± 0.14) and AC (1.54 ± 0.17) in exon 2, AA (1.22 ± 0.32), BC (1.72 ± 0.13), DD (1.67 ± 0.35), and CF (1.22 ± 0.45) in exon 3, and AD (2.35 ± 0.25) in exon 4 were significantly higher than those for the other genotypes (P ≤ 0.05) in DQA exons. There were 14 reconstructed haplotypes in the 3 exons from 290 individuals and Hap12 may be the diarrhea-resistant gene. Haplotype distribution was extremely uneven, and the SLA-DQA gene showed genetic linkage. In this study, we identified molecular genetic markers and provided a theoretical foundation for future pig anti-disease resistance breeding.

Antigenic Variation of East/Central/South African and Asian Chikungunya Virus Genotypes in Neutralization by Immune Sera.

PubMed

Chua, Chong-Long; Sam, I-Ching; Merits, Andres; Chan, Yoke-Fun

2016-08-01

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood. We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008-2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes. Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays.

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.

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

Accumulation of human T lymphotropic virus (HTLV)-I-specific T cell clones in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

PubMed

Höger, T A; Jacobson, S; Kawanishi, T; Kato, T; Nishioka, K; Yamamoto, K

1997-08-15

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraperesis (HAM/TSP) is a slowly progressive neurologic disorder following infection with HTLV-I. It is characterized by spasticity and hyper-reflexia of the lower extremities, urinary bladder disturbance, lower extremity muscle weakness, and sensory disturbances. HTLV-I, as an inducer of a strong humoral and cytotoxic response, is a well-known pathogenic factor for the progression of HAM/TSP. Peptides derived from proviral tax and env genes provide epitopes recognized by T cells. We herein report an accumulation of distinct clonotypes of alpha/beta TCR+ peripheral blood T lymphocytes from HAM/TSP patients in comparison with that observed in both asymptomatic carriers and healthy controls, using the reverse-transcriptase PCR/single-strand conformation polymorphism method. We also found that some of the accumulated T cell clones in the peripheral blood and cerebrospinal fluid are HTLV-I Tax(11-19) peptide specific. Such clones were found to expand strongly after being cultured with an HTLV-I Tax(11-19) peptide. Moreover, the cultured samples exhibited a strong MHC class I-restricted cytotoxic activity against HTLV-I Tax(11-19) peptide-expressing targets, and therefore most likely also include the disease-associated T cell clones observed in the patients. This is the first report of a direct assessment of Ag-specific T cell responses in fresh PBL and cerebrospinal fluid.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

PubMed

Zheng, Xuelian; Yang, Shixin; Zhang, Dengwei; Zhong, Zhaohui; Tang, Xu; Deng, Kejun; Zhou, Jianping; Qi, Yiping; Zhang, Yong

2016-07-01

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

Selenoglycosides in silico: ab initio-derived reparameterization of MM4, conformational analysis using histo-blood group ABH antigens and lectin docking as indication for potential of bioactivity

NASA Astrophysics Data System (ADS)

Strino, Francesco; Lii, Jenn-Huei; Koppisetty, Chaitanya A. K.; Nyholm, Per-Georg; Gabius, Hans-Joachim

2010-12-01

The identification of glycan epitopes such as the histo-blood group ABH determinants as docking sites for bacterial/viral infections and signals in growth regulation fuels the interest to develop non-hydrolysable mimetics for therapeutic applications. Inevitably, the required substitution of the linkage oxygen atom will alter the derivative's topology. Our study addresses the question of the impact of substitution of oxygen by selenium. In order to characterize spatial parameters and flexibility of selenoglycosides, we first performed ab initio calculations on model compounds to refine the MM4 force field. The following application of the resulting MM4R version appears to reduce the difference to ab initio data when compared to using the MM4 estimator. Systematic conformational searches on the derivatives of histo-blood group ABH antigens revealed increased flexibility with acquisition of additional low-energy conformer(s), akin to the behavior of S-glycosides. Docking analysis using the Glide program for eight test cases indicated potential for bioactivity, giving further experimental investigation a clear direction to testing Se-glycosides as lectin ligands.

Discovery of black dye crystal structure polymorphs: Implications for dye conformational variation in dye-sensitized solar cells

DOE PAGES

Cole, Jacqueline M.; Low, Kian Sing; Gong, Yun

2015-11-24

Here, we present the discovery of a new crystal structure polymorph (1) and pseudopolymorph (2) of the Black Dye, one of the world’s leading dyes for dye-sensitized solar cells, DSSCs (10.4% device performance efficiency). This reveals that Black Dye molecules can adopt multiple low-energy conformers. This is significant since it challenges existing models of the Black Dye···TiO 2 adsorption process that renders a DSSC working electrode; these have assumed a single molecular conformation that refers to the previously reported Black Dye crystal structure (3). The marked structural differences observed between 1, 2, and 3 make the need for modeling multiplemore » conformations more acute. Additionally, the ordered form of the Black Dye (1) provides a more appropriate depiction of its anionic structure, especially regarding its anchoring group and NCS bonding descriptions. The tendency toward NCS ligand isomerism, evidenced via the disordered form 2, has consequences for electron injection and electron recombination in Black Dye embedded DSSC devices. Dyes 2 and 3 differ primarily by the absence or presence of a solvent of crystallization, respectively; solvent environment effects on the dye are thereby elucidated. This discovery of multiple Black Dye conformers from diffraction, with atomic-level definition, complements recently reported nanoscopic evidence for multiple dye conformations existing at a dye···TiO 2 interface, for a chemically similar DSSC dye; those results emanated from imaging and spectroscopy, but were unresolved at the submolecular level. Taken together, these findings lead to the general notion that multiple dye conformations should be explicitly considered when modeling dye···TiO 2 interfaces in DSSCs, at least for ruthenium-based dye complexes.« less

Application of Solid-State NMR to Reveal Structural Differences in Cefazolin Sodium Pentahydrate from Different Manufacturing Processes

NASA Astrophysics Data System (ADS)

Tian, Ye; Wang, Wei D.; Zou, Wen-Bo; Qian, Jian-Qin; Hu, Chang-Qin

2018-04-01

The solid form of an active pharmaceutical ingredient is important when developing a new chemical entity. A solid understanding of the crystal structure and morphology that affect the mechanical and physical characteristics of pharmaceutical powders determines the manufacturing process. Solid-state NMR, thermogravimetric analysis, X-ray diffraction, and Fourier-transform infrared spectroscopy were combined with theoretical calculation to investigate different crystal packings of α-cefazolin sodium from three different vendors and conformational polymorphism was identified to exist in the α-cefazolin sodium. Marginal differences observed among CEZ-Na pentahydrate 1, 2, and 3 were speculated as the proportion of conformation 2. Understanding the differences in the polymorphic structure of α-cefazolin sodium may help with making modifications to incorporate new knowledge with a product’s development.

The inverse podant [Li3(NBut)3S)]+ stabilises a single ethylene oxide OCH=CH2 anion as a high- and low-temperature polymorph of [(thf)3Li3(OCH=CH2)(NBut)3S)].

PubMed

Walfort, B; Pandey, S K; Stalke, D

2001-09-07

A single ethylene oxide anion derived from the ether cleavage reaction of thf with ButLi is stabilised by the inverse podant [Li3(NBut)3S)]+ to give a high- and a low-temperature polymorph with a considerable difference in conformation and packing.

Collapsed state of polyglutamic acid results in amyloid spherulite formation

PubMed Central

Stehli, Daniel; Mulaj, Mentor; Miti, Tatiana; Traina, Joshua; Foley, Joseph; Muschol, Martin

2015-01-01

Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly. PMID:28232889

Collapsed state of polyglutamic acid results in amyloid spherulite formation.

PubMed

Stehli, Daniel; Mulaj, Mentor; Miti, Tatiana; Traina, Joshua; Foley, Joseph; Muschol, Martin

2015-01-01

Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly.

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

PubMed Central

Ringe, Rajesh P.; Sanders, Rogier W.; Yasmeen, Anila; Kim, Helen J.; Lee, Jeong Hyun; Cupo, Albert; Korzun, Jacob; Derking, Ronald; van Montfort, Thijs; Julien, Jean-Philippe; Wilson, Ian A.; Klasse, Per Johan; Ward, Andrew B.; Moore, John P.

2013-01-01

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated. PMID:24145402

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

PubMed Central

Irving, James A.; Miranda, Elena; Haq, Imran; Perez, Juan; Kotov, Vadim R.; Faull, Sarah V.; Motamedi-Shad, Neda; Lomas, David A.

2015-01-01

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen–whose native state is susceptible to the formation of a proto-oligomeric intermediate–we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states. PMID:25738741

Reassessing the Detection of B-Virus–Specific Serum Antibodies

PubMed Central

Katz, David; Shi, Wei; Wildes, Martin J; Krug, Peter W; Hilliard, Julia K

2012-01-01

B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers. PMID:23561886

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

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

Kong, Rui; Xu, Kai; Zhou, Tongqing

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

Structural and functional characterization of the hazelnut allergen Cor a 8

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

Offermann, Lesa R.; Bublin, Merima; Perdue, Makenzie L.

Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious,more » which may contribute to limited cross-reactivity between peach and hazelnut allergens. The differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.« less

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

Structural and functional characterization of the hazelnut allergen Cor a 8

DOE PAGES

Offermann, Lesa R.; Bublin, Merima; Perdue, Makenzie L.; ...

2015-09-28

Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious,more » which may contribute to limited cross-reactivity between peach and hazelnut allergens. The differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.« less

The amyloid fold of Gad m 1 epitopes governs IgE binding

PubMed Central

Sánchez, Rosa; Martínez, Javier; Castro, Ana; Pedrosa, María; Quirce, Santiago; Rodríguez-Pérez, Rosa; Gasset, María

2016-01-01

Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. PMID:27597317

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

DOE PAGES

Kong, Rui; Xu, Kai; Zhou, Tongqing; ...

2016-05-13

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

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.

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

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

Polymorphic transformation of helical flagella of bacteria

NASA Astrophysics Data System (ADS)

Lim, Sookkyung; Howard Berg Collaboration; William Ko Collaboration; Yongsam Kim Collaboration; Wanho Lee Collaboration; Charles Peskin Collaboration

2016-11-01

Bacteria such as E. coli swim in an aqueous environment by utilizing the rotation of flagellar motors and alternate two modes of motility, runs and tumbles. Runs are steady forward swimming driven by bundles of flagellar filaments whose motors are turning CCW; tumbles involve a reorientation of the direction of swimming triggered by motor reversals. During tumbling, the helical flagellum undergoes polymorphic transformations, which is a local change in helical pitch, helical radius, and handedness. In this work, we investigate the underlying mechanism of structural conformation and how this polymorphic transition plays a role in bacterial swimming. National Science Foundation.

Associations of polymorphisms in the Pit-1 gene with growth and carcass traits in Angus beef cattle.

PubMed

Zhao, Q; Davis, M E; Hines, H C

2004-08-01

The Pit-1 gene was studied as a candidate for genetic markers of growth and carcass traits. Angus beef cattle that were divergently selected for high- or low-blood serum IGF-I concentration were used in this study. The single-strand conformation polymorphism method was used to identify polymorphism in the Pit-1 gene including regions from intron 2 to exon 6. Two polymorphisms, Pit1I3H (HinfI) and Pit1I3NL (NlaIII), were detected in intron 3 of the Pit-1 gene. One polymorphism, Pit1I4N (BstNI), was found in intron 4, and a single nucleotide polymorphism, Pit1I5, was found in intron 5. The previously reported polymorphism in exon 6, Pit1E6H (HinfI), was also studied in 416 Angus beef cattle. Associations of the polymorphisms with growth traits, carcass traits, and IGF-I concentration were analyzed using a general linear model procedure. No significant associations were observed between these polymorphisms and growth and carcass traits.

A general protocol for the generation of Nanobodies for structural biology

PubMed Central

Pardon, Els; Laeremans, Toon; Triest, Sarah; Rasmussen, Søren G. F.; Wohlkönig, Alexandre; Ruf, Armin; Muyldermans, Serge; Hol, Wim G. J.; Kobilka, Brian K.; Steyaert, Jan

2015-01-01

There is growing interest in using antibodies as auxiliary proteins to crystallize proteins. Here, we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has the competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, enabling to solve the structures of the most challenging proteins by Nanobody-assisted X-ray crystallography in a time span of 6 to 12 months. PMID:24577359

Study on four polymorphs of bifendate based on X-ray crystallography.

PubMed

Nie, Jinju; Yang, Dezhi; Hu, Kun; Lu, Yang

2016-05-01

Bifendate, a synthetic anti-hepatitis drug, exhibits polycrystalline mode phenomena with 2 polymorphs reported (forms A and B). Single crystals of the known crystalline form B and 3 new crystallosolvates involving bifendate solvated with tetrahydrofuran (C), dioxane (D), and pyridine (E) in a stoichiometric ratio of 1:1 were obtained and characterized by X-ray crystallography, thermal analysis, and Fourier transform infrared (FT-IR) spectroscopy. The differences in molecular conformation, intermolecular interaction and crystal packing arrangement for the four polymorphs were determined and the basis for the polymorphisms was investigated. The rotation of single bonds resulted in different orientations for the biphenyl, methyl ester and methoxyl groups. All guest solvent molecules interacted with the host molecule via an interesting intercalative mode along the [1 0 0] direction in the channel formed by the host molecules through weak aromatic stacking interactions or non-classical hydrogen bonds, of which the volume and planarity played an important role in the intercalation of the host with the guest. The incorporation of solvent-augmented rotation of the C-C bond of the biphenyl group had a striking effect on the host molecular conformation and contributed to the formation of bifendate polymorphs. Moreover, the simulated powder X-ray diffraction (PXRD) patterns for each form were calculated on the basis of the single-crystal data and proved to be unique. The single-crystal structures of the four crystalline forms are reported in this paper.

Structural polymorphism of a cytosine-rich DNA sequence forming i-motif structure: Exploring pH based biosensors.

PubMed

Ahmed, Saami; Kaushik, Mahima; Chaudhary, Swati; Kukreti, Shrikant

2018-05-01

Sequence recognition and conformational polymorphism enable DNA to emerge out as a substantial tool in fabricating the devices within nano-dimensions. These DNA associated nano devices work on the principle of conformational switches, which can be facilitated by many factors like sequence of DNA/RNA strand, change in pH or temperature, enzyme or ligand interactions etc. Thus, controlling these DNA conformational changes to acquire the desired function is significant for evolving DNA hybridization biosensor, used in genetic screening and molecular diagnosis. For exploring this conformational switching ability of cytosine-rich DNA oligonucleotides as a function of pH for their potential usage as biosensors, this study has been designed. A C-rich stretch of DNA sequence (5'-TCCCCCAATTAATTCCCCCA-3'; SG20c) has been investigated using UV-Thermal denaturation, poly-acrylamide gel electrophoresis and CD spectroscopy. The SG20c sequence is shown to adopt various topologies of i-motif structure at low pH. This pH dependent transition of SG20c from unstructured single strand to unimolecular and bimolecular i-motif structures can further be exploited for its utilization as switching on/off pH-based biosensors. Copyright © 2018. Published by Elsevier B.V.

Protein conformation and disease : pathological consequences of analogous mutations in homologous proteins.

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

Stevens, F. J.; Pokkuluri, P. R.; Schiffer, M.

2000-12-19

The antibody light chain variable domain (V{sub L}){sup 1} and myelin protein zero (MPZ) are representatives of the functionally diverse immunoglobulin superfamily. The V{sub L} is a subunit of the antigen-binding component of antibodies, while MPZ is the major membrane-linked constituent of the myelin sheaths that coat peripheral nerves. Despite limited amino acid sequence homology, the conformations of the core structures of the two proteins are largely superimposable. Amino acid variations in V{sub L} account for various conformational disease outcomes, including amyloidosis. However, the specific amino acid changes in V{sub L} that are responsible for disease have been obscured bymore » multiple concurrent primary structure alterations. Recently, certain demyelination disorders have been linked to point mutations and single amino acid polymorphisms in MPZ. We demonstrate here that some pathogenic variations in MPZ correspond to changes suspected of determining amyloidosis in V{sub L}. This unanticipated observation suggests that studies of the biophysical origin of conformational disease in one member of a superfamily of homologous proteins may have implications throughout the superfamily. In some cases, findings may account for overt disease; in other cases, due to the natural repertoire of inherited polymorphisms, variations in a representative protein may predict subclinical impairment of homologous proteins.« less

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

PubMed

Tournamille, Christophe; Filipe, Anne; Wasniowska, Kazimiera; Gane, Pierre; Lisowska, Elwira; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

2003-09-01

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

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

B-Cell Epitopes in GroEL of Francisella tularensis

PubMed Central

Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

2014-01-01

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: the case of casein micelle.

PubMed

Johansson, Annette; Lugand, Damien; Rolet-Répécaud, Odile; Mollé, Daniel; Delage, Marie-Madeleine; Peltre, Gabriel; Marchesseau, Sylvie; Léonil, Joëlle; Dupont, Didier

2009-03-01

In milk, kappa-, beta-, alphas(1)- and alphas(2)-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although kappa-CN and alphas(2)-CN are minor proteins in the micelle (ratio 1:1:4:4 for kappa, alphas(2), alphas(1), beta) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for alphas(1)-CN and beta-CN, respectively. Most of the beta-CN and kappa-CN epitopes were identified, while about 50% of alphas(1)-CN and alphas(2)-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with alphas(1)-CN. The C-terminal glycomacropeptide of kappa-CN and the C-terminal moiety of beta-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of alphas(2) were alphas(2)-CN (f96-114) and (f16-35). Cross-reaction between alphas(2)-CN specific antibodies with alphas(1)-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure.

Dengue virus-like particles mimic the antigenic properties of the infectious dengue virus envelope.

PubMed

Metz, Stefan W; Thomas, Ashlie; White, Laura; Stoops, Mark; Corten, Markus; Hannemann, Holger; de Silva, Aravinda M

2018-04-02

The 4 dengue serotypes (DENV) are mosquito-borne pathogens that are associated with severe hemorrhagic disease. DENV particles have a lipid bilayer envelope that anchors two membrane glycoproteins prM and E. Two E-protein monomers form head-to-tail homodimers and three E-dimers align to form "rafts" that cover the viral surface. Some human antibodies that strongly neutralize DENV bind to quaternary structure epitopes displayed on E protein dimers or higher order structures forming the infectious virus. Expression of prM and E in cell culture leads to the formation of DENV virus-like particles (VLPs) which are smaller than wildtype virus particles and replication defective due to the absence of a viral genome. There is no data available that describes the antigenic landscape on the surface of flavivirus VLPs in comparison to the better studied infectious virion. A large panel of well characterized antibodies that recognize epitope of ranging complexity were used in biochemical analytics to obtain a comparative antigenic surface view of VLPs in respect to virus particles. DENV patient serum depletions were performed the show the potential of VLPs in serological diagnostics. VLPs were confirmed to be heterogeneous in size morphology and maturation state. Yet, we show that many highly conformational and quaternary structure-dependent antibody epitopes found on virus particles are efficiently displayed on DENV1-4 VLP surfaces as well. Additionally, DENV VLPs can efficiently be used as antigens to deplete DENV patient sera from serotype specific antibody populations. This study aids in further understanding epitopic landscape of DENV VLPs and presents a comparative antigenic surface view of VLPs in respect to virus particles. We propose the use VLPs as a safe and practical alternative to infectious virus as a vaccine and diagnostic antigen.

Structural Analysis of Der p 1–Antibody Complexes and Comparison with Complexes of Proteins or Peptides with Monoclonal Antibodies

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

Osinski, Tomasz; Pomés, Anna; Majorek, Karolina A.

Der p 1 is a major allergen from the house dust mite, Dermatophagoides pteronyssinus, that belongs to the papain-like cysteine protease family. To investigate the antigenic determinants of Der p 1, we determined two crystal structures of Der p 1 in complex with the Fab fragments of mAbs 5H8 or 10B9. Epitopes for these two Der p 1–specific Abs are located in different, nonoverlapping parts of the Der p 1 molecule. Nevertheless, surface area and identity of the amino acid residues involved in hydrogen bonds between allergen and Ab are similar. The epitope for mAb 10B9 only showed a partialmore » overlap with the previously reported epitope for mAb 4C1, a cross-reactive mAb that binds Der p 1 and its homolog Der f 1 from Dermatophagoides farinae. Upon binding to Der p 1, the Fab fragment of mAb 10B9 was found to form a very rare α helix in its third CDR of the H chain. To provide an overview of the surface properties of the interfaces formed by the complexes of Der p 1–10B9 and Der p 1–5H8, along with the complexes of 4C1 with Der p 1 and Der f 1, a broad analysis of the surfaces and hydrogen bonds of all complexes of Fab–protein or Fab–peptide was performed. This work provides detailed insight into the cross-reactive and specific allergen–Ab interactions in group 1 mite allergens. The surface data of Fab–protein and Fab–peptide interfaces can be used in the design of conformational epitopes with reduced Ab binding for immunotherapy.« less

Identification of small molecules capable of regulating conformational changes of telomeric G-quadruplex

NASA Astrophysics Data System (ADS)

Chen, Shuo-Bin; Liu, Guo-Cai; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

2018-02-01

Design of small molecules targeted at human telomeric G-quadruplex DNA is an extremely active research area. Interestingly, the telomeric G-quadruplex is a highly polymorphic structure. Changes in its conformation upon small molecule binding may be a powerful method to achieve a desired biological effect. However, the rational development of small molecules capable of regulating conformational change of telomeric G-quadruplex structures is still challenging. In this study, we developed a reliable ligand-based pharmacophore model based on isaindigotone derivatives with conformational change activity toward telomeric G-quadruplex DNA. Furthermore, virtual screening of database was conducted using this pharmacophore model and benzopyranopyrimidine derivatives in the database were identified as a strong inducer of the telomeric G-quadruplex DNA conformation, transforming it from hybrid-type structure to parallel structure.

Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa.

PubMed

Hölzemer, Angelique; Thobakgale, Christina F; Jimenez Cruz, Camilo A; Garcia-Beltran, Wilfredo F; Carlson, Jonathan M; van Teijlingen, Nienke H; Mann, Jaclyn K; Jaggernath, Manjeetha; Kang, Seung-gu; Körner, Christian; Chung, Amy W; Schafer, Jamie L; Evans, David T; Alter, Galit; Walker, Bruce D; Goulder, Philip J; Carrington, Mary; Hartmann, Pia; Pertel, Thomas; Zhou, Ruhong; Ndung'u, Thumbi; Altfeld, Marcus

2015-11-01

Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I-presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.

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

Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. Fc{gamma}RIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of Fc{gamma}RIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of Fc{gamma}RIIa (Fc{gamma}RIIa-HR) and the Fc region of amore » humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence Fc{gamma}RIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for Fc{gamma}RIIa (IV.3), Fc{gamma}RIIb (X63-21), and a pan Fc{gamma}RII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of Fc{gamma}RIIa and Fc{gamma}RIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of Fc{gamma}RIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.« less

A comparative study of two polymorphs of L-aspartic acid hydrochloride.

PubMed

Benali-Cherif, Rim; Takouachet, Radhwane; Bendeif, El-Eulmi; Benali-Cherif, Nourredine

2014-07-01

Two polymorphs of L-aspartic acid hydrochloride, C4H8NO4(+)·Cl(-), were obtained from the same aqueous solution. Their crystal structures have been determined from single-crystal data collected at 100 K. The crystal structures revealed three- and two-dimensional hydrogen-bonding networks for the triclinic and orthorhombic polymorphs, respectively. The cations and anions are connected to one another via N-H···Cl and O-H···Cl interactions and form alternating cation-anion layer-like structures. The two polymorphs share common structural features; however, the conformations of the L-aspartate cations and the crystal packings are different. Furthermore, the molecular packing of the orthorhombic polymorph contains more interesting interactions which seems to be a favourable factor for more efficient charge transfer within the crystal.

Thermal stability of self-assembled peptide vaccine materials.

PubMed

Sun, Tao; Han, Huifang; Hudalla, Gregory A; Wen, Yi; Pompano, Rebecca R; Collier, Joel H

2016-01-01

The majority of current vaccines depend on a continuous "cold chain" of storage and handling between 2 and 8°C. Vaccines experiencing temperature excursions outside this range can suffer from reduced potency. This thermal sensitivity results in significant losses of vaccine material each year and risks the administration of vaccines with diminished protective ability, issues that are heightened in the developing world. Here, using peptide self-assemblies based on the fibril-forming peptide Q11 and containing the epitopes OVA323-339 from ovalbumin or ESAT651-70 from Mycobacterium tuberculosis, the chemical, conformational, and immunological stability of supramolecular peptide materials were investigated. It was expected that these materials would exhibit advantageous thermal stability owing to their adjuvant-free and fully synthetic construction. Neither chemical nor conformational changes were observed for either peptide when stored at 45°C for 7days. ESAT651-70-Q11 was strongly immunogenic whether it was stored as a dry powder or as aqueous nanofibers, showing undiminished immunogenicity even when stored as long as six months at 45°C. This result was in contrast to ESAT651-70 conjugated to a protein carrier and adjuvanted with alum, which demonstrated marked thermal sensitivity in these conditions. Antibody titers and affinities were undiminished in mice for OVA323-339-Q11 if it was stored as assembled nanofibers, yet some diminishment was observed for material stored as a dry powder. The OVA study was done in a different mouse strain and with a different prime/boost regimen, and so it should not be compared directly with the study for the ESAT epitope. This work indicates that peptide self-assemblies can possess attractive thermal stability properties in the context of vaccine development. Almost all current vaccines must be maintained within a tight and refrigerated temperature range, usually between 2 and 8°C. This presents significant challenges for their distribution, especially in the developing world. Here we report on the surprisingly robust thermal stability of a self-assembled peptide vaccine. In particular a self-assembled peptide vaccine containing a tuberculosis epitope maintained all of its potency in mice when exposed to an extreme thermal treatment of six months at 45°C. In a different mouse model, we investigated another model epitope and found some storage conditions where potency was diminished. Overall this study illustrates that some self-assembled peptide vaccines can have remarkable thermal stability. Copyright © 2015 Acta Materialia Inc. Published by 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.

Anti-LC1 autoantibodies in patients with chronic hepatitis C virus infection.

PubMed

Béland, Kathie; Lapierre, Pascal; Marceau, Gabriel; Alvarez, Fernando

2004-03-01

Various autoantibodies have been reported in patients chronically infected by hepatitis C virus. 2% to 10% of theses patients have anti-liver-kidney microsome type 1 (anti-LKM1) autoantibodies. In type 2 autoimmune hepatitis, anti-LKM1 autoantibodies are frequently associated with anti-liver-cytosol type 1 (anti-LC1) autoantibodies. To determine the prevalence of anti-LC1 autoantibodies in a hepatitis C-positive population and characterize their reactivity. 146 patients suffering from liver diseases, of which 99 were chronically infected by hepatitis C virus, were tested by Western blotting and immunoprecipitation to detect and characterize anti-LC1 autoantibodies. 12% of this hepatitis C population had anti-LC1 autoantibodies. LC1 positivity by Western blotting was 30% of LC1+ sera. Epitopes were found throughout the protein but linear epitopes were situated in the 395-541 amino acid region of formiminotransferase cyclodeaminase. Three putative conformational epitopes were identified by phage display. Anti-LC1 autoantibodies are as prevalent as anti-LKM1 autoantibodies in patients infected with hepatitis C virus and their production is not dependent of anti-LKM1 autoantibodies formation. Autoantibody reactivity against the anti-LC1 antigen is different in hepatitis C than in type 2 autoimmune hepatitis. Anti-LC1 autoantibodies can now be regarded as a serological marker of autoimmunity in chronic hepatitis C infection.

Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

PubMed

Yang, Yang; Chen, Zhong-Wei; Hurlburt, Barry K; Li, Gui-Ling; Zhang, Yong-Xia; Fei, Dan-Xia; Shen, Hai-Wang; Cao, Min-Jie; Liu, Guang-Ming

2017-05-01

Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A 71 G 74 S 69 D 75 T 73 F 72 V 67 ) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Glutathione exposes sequential IgE-epitopes in ovomucoid relevant in persistent egg allergy.

PubMed

Roth-Walter, Franziska; Starkl, Philipp; Zuberbier, Torsten; Hummel, Karin; Nöbauer, Karin; Razzazi-Fazeli, Ebrahim; Brunner, Richard; Pali-Schöll, Isabella; Kinkel, Janis; Felix, Ferdinand; Jensen-Jarolim, Erika; Kinaciyan, Tamar

2013-03-01

Patients with persistent egg allergy have more immunoglobulin E (IgE) against sequential than conformational epitopes of ovomucoid (OVO). Here, we aimed to identify compounds capable to render sequential epitopes in egg. Glutathione was used for in vitro reduction of OVO and circular dichroism analyses were performed. Glutathione reduced OVO in a concentration-dependent manner. Egg white was analyzed for reduced proteins with a thiol probe and by MALDI-TOF/TOF. In unprocessed total egg white, several reduced proteins were detected by the thiol probe, among them reduced ovalbumin could be confirmed with MS analyses. Egg-allergics or sensitized controls were tested serologically (n = 19) for IgE against native and reduced OVO and in skin prick tests (n = 9). More patients had IgE against reduced than native OVO in Western blots. In skin prick test, five out of seven persistent egg-allergics and none of the controls reacted with reduced OVO. Reduced egg proteins are present in natural egg white. Glutathione, which is present in egg and furthermore is used as texture-improving additive in processed food, is capable of reducing OVO. Patients with persistent egg allergy reacted rather to reduce the native OVO. Hence, our data indicate that reduction is a novel natural and processing-associated principle, which contributes to the allergenicity of food. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prediction of merozoite surface protein 1 and apical membrane antigen 1 vaccine efficacies against Plasmodium chabaudi malaria based on prechallenge antibody responses.

PubMed

Lynch, Michelle M; Cernetich-Ott, Amy; Weidanz, William P; Burns, James M

2009-03-01

For the development of blood-stage malaria vaccines, there is a clear need to establish in vitro measures of the antibody-mediated and the cell-mediated immune responses that correlate with protection. In this study, we focused on establishing correlates of antibody-mediated immunity induced by immunization with apical membrane antigen 1 (AMA1) and merozoite surface protein 1(42) (MSP1(42)) subunit vaccines. To do so, we exploited the Plasmodium chabaudi rodent model, with which we can immunize animals with both protective and nonprotective vaccine formulations and allow the parasitemia in the challenged animals to peak. Vaccine formulations were varied with regard to the antigen dose, the antigen conformation, and the adjuvant used. Prechallenge antibody responses were evaluated by enzyme-linked immunosorbent assay and were tested for a correlation with protection against nonlethal P. chabaudi malaria, as measured by a reduction in the peak level of parasitemia. The analysis showed that neither the isotype profile nor the avidity of vaccine-induced antibodies correlated with protective efficacy. However, high titers of antibodies directed against conformation-independent epitopes were associated with poor vaccine performance and may limit the effectiveness of protective antibodies that recognize conformation-dependent epitopes. We were able to predict the efficacies of the P. chabaudi AMA1 (PcAMA1) and P. chabaudi MSP1(42) (PcMSP1(42)) vaccines only when the prechallenge antibody titers to both refolded and reduced/alkylated antigens were considered in combination. The relative importance of these two measures of vaccine-induced responses as predictors of protection differed somewhat for the PcAMA1 and the PcMSP1(42) vaccines, a finding confirmed in our final immunization and challenge study. A similar approach to the evaluation of vaccine-induced antibody responses may be useful during clinical trials of Plasmodium falciparum AMA1 and MSP1(42) vaccines.

Prediction of Merozoite Surface Protein 1 and Apical Membrane Antigen 1 Vaccine Efficacies against Plasmodium chabaudi Malaria Based on Prechallenge Antibody Responses▿

PubMed Central

Lynch, Michelle M.; Cernetich-Ott, Amy; Weidanz, William P.; Burns, James M.

2009-01-01

For the development of blood-stage malaria vaccines, there is a clear need to establish in vitro measures of the antibody-mediated and the cell-mediated immune responses that correlate with protection. In this study, we focused on establishing correlates of antibody-mediated immunity induced by immunization with apical membrane antigen 1 (AMA1) and merozoite surface protein 142 (MSP142) subunit vaccines. To do so, we exploited the Plasmodium chabaudi rodent model, with which we can immunize animals with both protective and nonprotective vaccine formulations and allow the parasitemia in the challenged animals to peak. Vaccine formulations were varied with regard to the antigen dose, the antigen conformation, and the adjuvant used. Prechallenge antibody responses were evaluated by enzyme-linked immunosorbent assay and were tested for a correlation with protection against nonlethal P. chabaudi malaria, as measured by a reduction in the peak level of parasitemia. The analysis showed that neither the isotype profile nor the avidity of vaccine-induced antibodies correlated with protective efficacy. However, high titers of antibodies directed against conformation-independent epitopes were associated with poor vaccine performance and may limit the effectiveness of protective antibodies that recognize conformation-dependent epitopes. We were able to predict the efficacies of the P. chabaudi AMA1 (PcAMA1) and P. chabaudi MSP142 (PcMSP142) vaccines only when the prechallenge antibody titers to both refolded and reduced/alkylated antigens were considered in combination. The relative importance of these two measures of vaccine-induced responses as predictors of protection differed somewhat for the PcAMA1 and the PcMSP142 vaccines, a finding confirmed in our final immunization and challenge study. A similar approach to the evaluation of vaccine-induced antibody responses may be useful during clinical trials of Plasmodium falciparum AMA1 and MSP142 vaccines. PMID:19116303

ACE phenotyping in human heart.

PubMed

Tikhomirova, Victoria E; Kost, Olga A; Kryukova, Olga V; Golukhova, Elena Z; Bulaeva, Naida I; Zholbaeva, Aigerim Z; Bokeria, Leo A; Garcia, Joe G N; Danilov, Sergei M

2017-01-01

Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs. We performed ACE phenotyping (ACE levels, conformation and kinetic characteristics) in the human heart and compared it with that in the lung. ACE activity in heart tissues was 10-15 lower than that in lung. Various ACE effectors, LMW endogenous ACE inhibitors and HMW ACE-binding partners, were shown to be present in both heart and lung tissues. "Conformational fingerprint" of heart ACE (i.e., the pattern of 17 mAbs binding to different epitopes on the ACE surface) significantly differed from that of lung ACE, which reflects differences in the local conformations of these ACEs, likely controlled by different ACE glycosylation in these organs. Substrate specificity and pH-optima of the heart and lung ACEs also differed. Moreover, even within heart the apparent ACE activities, the local ACE conformations, and the content of ACE inhibitors differ in atria and ventricles. Significant differences in the local conformations and kinetic properties of heart and lung ACEs demonstrate tissue specificity of ACE and provide a structural base for the development of mAbs able to distinguish heart and lung ACEs as a potential blood test for predicting atrial fibrillation risk.

A Delicate Interplay of Structure, Dynamics, and Thermodynamics for Function: A High Pressure NMR Study of Outer Surface Protein A

PubMed Central

Kitahara, Ryo; Simorellis, Alana K.; Hata, Kazumi; Maeno, Akihiro; Yokoyama, Shigeyuki; Koide, Shohei; Akasaka, Kazuyuki

2012-01-01

Outer surface protein A (OspA) is a crucial protein in the infection of Borrelia burgdorferi causing Lyme disease. We studied conformational fluctuations of OspA with high-pressure 15N/1H two-dimensional NMR along with high-pressure fluorescence spectroscopy. We found evidence within folded, native OspA for rapid local fluctuations of the polypeptide backbone in the nonglobular single layer β-sheet connecting the N- and C-terminal domains with τ << ms, which may give the two domains certain independence in mobility and thermodynamic stability. Furthermore, we found that folded, native OspA is in equilibrium (τ >> ms) with a minor conformer I, which is almost fully disordered and hydrated for the entire C-terminal part of the polypeptide chain from β8 to the C-terminus. Conformer I is characterized with ΔG0 = 32 ± 9 kJ/mol and ΔV0 = −140 ± 40 mL/mol, populating only ∼0.001% at 40°C at 0.1 MPa, pH 5.9. Because in the folded conformer the receptor binding epitope of OspA is buried in the C-terminal domain, its transition into conformer I under in vivo conditions may be critical for the infection of B. burgdorferi. The formation and stability of the peculiar conformer I are apparently supported by a large packing defect or cavity located in the C-terminal domain. PMID:22385863

Strategies in the development of vaccines to prevent infections with group A streptococcus

PubMed Central

Good, Michael F; Batzloff, Michael; Pandey, Manisha

2013-01-01

There has long been interest and demand for the development of a vaccine to prevent infections caused by the Gram-positive organism group A streptococcus. Despite numerous efforts utilizing advanced approaches such as genomics, proteomics and bio-informatics, there is currently no vaccine. Here we review various strategies employed to achieve this goal. We also discuss the approach that we have pursued, a non-host reactive, conformationally constrained minimal B cell epitope from within the C-repeat region of M-protein, and the potential limitations in moving forward. PMID:23863455

Structural and abinitio studies on the polymorphism of iminophosphorane (CH3C6H4)3Pdbnd NP[(dbnd O)(OPh)2

NASA Astrophysics Data System (ADS)

Petric, Mihaela F.; Crisan, Manuela E.; Chumakov, Yurii M.; Varga, Richard A.; Micle, Andreea; Neda, Ion; Ilia, Gheorghe

2015-03-01

Two polymorphic forms of a new iminophosphorane have been investigated by infrared, nuclear magnetic resonance and mass spectroscopy, X-ray crystallography and studied through ab initio quantum chemical calculations. The monoclinic polymorph α contains two independent molecules (αI and αII) in the asymmetric unit, while the orthorhombic polymorph ß has one molecule in the asymmetric unit. The molecules in polymorphs α and β adopt different conformations. Hirshfeld surfaces and fingerprint plots were generated in order to compare the two independent molecules αI and αII in the asymmetric unit and also for a comparison of ß molecule, in the orthorhombic crystal system, with the previously reported monoclinic polymorph. The results show that the packing motifs in polymorphs α and β differ mainly due to the redistribution of Csbnd H⋯O and Csbnd H⋯π hydrogen-bond interactions rather than their percentage Hirshfeld surface area contributions. The dipole-dipole interactions significantly influence the intermolecular interactions in polymorphs α and β. The calculated lattice energies indicate that polymorph α is slightly more stable than polymorph α.

A Conformational Change of C Fragment of Tetanus Neurotoxin Reduces Its Ganglioside-Binding Activity but Does Not Destroy Its Immunogenicity ▿

PubMed Central

Yu, Rui; Yi, Shaoqiong; Yu, Changming; Fang, Ting; Liu, Shuling; Yu, Ting; Song, Xiaohong; Fu, Ling; Hou, Lihua; Chen, Wei

2011-01-01

The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside GT1b and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects. PMID:21813664

Large-Scale Conformational Changes of Trypanosoma cruzi Proline Racemase Predicted by Accelerated Molecular Dynamics Simulation

PubMed Central

McCammon, J. Andrew

2011-01-01

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life-threatening illness affecting 11–18 million people. Currently available treatments are limited, with unacceptable efficacy and safety profiles. Recent studies have revealed an essential T. cruzi proline racemase enzyme (TcPR) as an attractive candidate for improved chemotherapeutic intervention. Conformational changes associated with substrate binding to TcPR are believed to expose critical residues that elicit a host mitogenic B-cell response, a process contributing to parasite persistence and immune system evasion. Characterization of the conformational states of TcPR requires access to long-time-scale motions that are currently inaccessible by standard molecular dynamics simulations. Here we describe advanced accelerated molecular dynamics that extend the effective simulation time and capture large-scale motions of functional relevance. Conservation and fragment mapping analyses identified potential conformational epitopes located in the vicinity of newly identified transient binding pockets. The newly identified open TcPR conformations revealed by this study along with knowledge of the closed to open interconversion mechanism advances our understanding of TcPR function. The results and the strategy adopted in this work constitute an important step toward the rationalization of the molecular basis behind the mitogenic B-cell response of TcPR and provide new insights for future structure-based drug discovery. PMID:22022240

Large-scale conformational changes of Trypanosoma cruzi proline racemase predicted by accelerated molecular dynamics simulation.

PubMed

de Oliveira, César Augusto F; Grant, Barry J; Zhou, Michelle; McCammon, J Andrew

2011-10-01

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life-threatening illness affecting 11-18 million people. Currently available treatments are limited, with unacceptable efficacy and safety profiles. Recent studies have revealed an essential T. cruzi proline racemase enzyme (TcPR) as an attractive candidate for improved chemotherapeutic intervention. Conformational changes associated with substrate binding to TcPR are believed to expose critical residues that elicit a host mitogenic B-cell response, a process contributing to parasite persistence and immune system evasion. Characterization of the conformational states of TcPR requires access to long-time-scale motions that are currently inaccessible by standard molecular dynamics simulations. Here we describe advanced accelerated molecular dynamics that extend the effective simulation time and capture large-scale motions of functional relevance. Conservation and fragment mapping analyses identified potential conformational epitopes located in the vicinity of newly identified transient binding pockets. The newly identified open TcPR conformations revealed by this study along with knowledge of the closed to open interconversion mechanism advances our understanding of TcPR function. The results and the strategy adopted in this work constitute an important step toward the rationalization of the molecular basis behind the mitogenic B-cell response of TcPR and provide new insights for future structure-based drug discovery.

A Malaria Vaccine Based on the Polymorphic Block 2 Region of MSP-1 that Elicits a Broad Serotype-Spanning Immune Response

PubMed Central

Cowan, Graeme J. M.; Creasey, Alison M.; Dhanasarnsombut, Kelwalin; Thomas, Alan W.; Remarque, Edmond J.; Cavanagh, David R.

2011-01-01

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen. PMID:22073118

Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

2018-01-01

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

Conformational dynamics underlie the activity of the auxin-binding protein, Nt-abp1.

PubMed

David, K; Carnero-Diaz, E; Leblanc, N; Monestiez, M; Grosclaude, J; Perrot-Rechenmann, C

2001-09-14

The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.

BST-2 Expression Modulates Small CD4-Mimetic Sensitization of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

PubMed Central

Prévost, Jérémie; von Bredow, Benjamin; Ding, Shilei; Brassard, Nathalie; Medjahed, Halima; Coutu, Mathieu; Melillo, Bruno; Bibollet-Ruche, Frédéric; Hahn, Beatrice H.; Kaufmann, Daniel E.; Smith, Amos B.; Sodroski, Joseph; Sauter, Daniel; Kirchhoff, Frank; Gee, Katrina; Neil, Stuart J.; Evans, David T.

2017-01-01

ABSTRACT Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals. IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication. PMID:28331088

Quantifying side-chain conformational variations in protein structure

PubMed Central

Miao, Zhichao; Cao, Yang

2016-01-01

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs. PMID:27845406

Quantifying side-chain conformational variations in protein structure

NASA Astrophysics Data System (ADS)

Miao, Zhichao; Cao, Yang

2016-11-01

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs.

Quantifying side-chain conformational variations in protein structure.

PubMed

Miao, Zhichao; Cao, Yang

2016-11-15

Protein side-chain conformation is closely related to their biological functions. The side-chain prediction is a key step in protein design, protein docking and structure optimization. However, side-chain polymorphism comprehensively exists in protein as various types and has been long overlooked by side-chain prediction. But such conformational variations have not been quantitatively studied and the correlations between these variations and residue features are vague. Here, we performed statistical analyses on large scale data sets and found that the side-chain conformational flexibility is closely related to the exposure to solvent, degree of freedom and hydrophilicity. These analyses allowed us to quantify different types of side-chain variabilities in PDB. The results underscore that protein side-chain conformation prediction is not a single-answer problem, leading us to reconsider the assessment approaches of side-chain prediction programs.

Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex E

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

Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β + T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides withmore » few restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.« less

Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex E

DOE PAGES

Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.; ...

2016-02-12

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β + T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides withmore » few restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.« less

Hybrid phage displaying SLAQVKYTSASSI induces protection against Candida albicans challenge in BALB/c mice.

PubMed

Wang, Yicun; Su, Quanping; Dong, Shuai; Shi, Hongxi; Gao, Xiang; Wang, Li

2014-01-01

The polymorphic fungus Candida albicans (C. albicans) can live as an aggressive pathogen and cause many diseases in hosts, for which no effective vaccine exists. The secreted aspartyl proteinase 2 (Sap2) plays a protective role in systemically infected BALB/c mice. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. Therefore, the emphasis is placed on developing new phage vaccine to inhibit C. albicans infection. In this study, the ability of the hybrid phage displaying the epitope SLAQVKYTSASSI and recombinant protein of Sap2 (rSap2) for inducing immune protective responses against C. albicans infection was evaluated by lymphoproliferative assay, to gather cytokine and antibody measurements in BALB/c mice. Our results showed that, strong cellular and humoral immune responses were induced in a mouse model immunized with hybrid phage or rSap2. Furthermore, the protection against lethal challenge with C. albicans was observed in mice vaccinated hybrid phage without adjuvant. These findings demonstrate that the hybrid phage displaying the epitope SLAQVKYTSASSI might be a potential vaccine against C. albicans infections.

Single strand conformation polymorphism based SNP and Indel markers for genetic mapping and synteny analysis of common bean (Phaseolus vulgaris L.)

PubMed Central

2009-01-01

Background Expressed sequence tags (ESTs) are an important source of gene-based markers such as those based on insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). Several gel based methods have been reported for the detection of sequence variants, however they have not been widely exploited in common bean, an important legume crop of the developing world. The objectives of this project were to develop and map EST based markers using analysis of single strand conformation polymorphisms (SSCPs), to create a transcript map for common bean and to compare synteny of the common bean map with sequenced chromosomes of other legumes. Results A set of 418 EST based amplicons were evaluated for parental polymorphisms using the SSCP technique and 26% of these presented a clear conformational or size polymorphism between Andean and Mesoamerican genotypes. The amplicon based markers were then used for genetic mapping with segregation analysis performed in the DOR364 × G19833 recombinant inbred line (RIL) population. A total of 118 new marker loci were placed into an integrated molecular map for common bean consisting of 288 markers. Of these, 218 were used for synteny analysis and 186 presented homology with segments of the soybean genome with an e-value lower than 7 × 10-12. The synteny analysis with soybean showed a mosaic pattern of syntenic blocks with most segments of any one common bean linkage group associated with two soybean chromosomes. The analysis with Medicago truncatula and Lotus japonicus presented fewer syntenic regions consistent with the more distant phylogenetic relationship between the galegoid and phaseoloid legumes. Conclusion The SSCP technique is a useful and inexpensive alternative to other SNP or Indel detection techniques for saturating the common bean genetic map with functional markers that may be useful in marker assisted selection. In addition, the genetic markers based on ESTs allowed the construction of a transcript map and given their high conservation between species allowed synteny comparisons to be made to sequenced genomes. This synteny analysis may support positional cloning of target genes in common bean through the use of genomic information from these other legumes. PMID:20030833

Further exploration of the conformational space of α-synuclein fibrils: solid-state NMR assignment of a high-pH polymorph.

PubMed

Verasdonck, Joeri; Bousset, Luc; Gath, Julia; Melki, Ronald; Böckmann, Anja; Meier, Beat H

2016-04-01

Polymorphism is a common and important phenomenon for protein fibrils which has been linked to the appearance of strains in prion and other neurodegenerative diseases. Parkinson disease is a frequently occurring neurodegenerative pathology, tightly associated with the formation of Lewy bodies. These deposits mainly consist of α-synuclein in fibrillar, β-sheet-rich form. α-synuclein is known to form numerous different polymorphs, which show distinct structural features. Here, we describe the chemical shift assignments, and derive the secondary structure, of a polymorph that was fibrillized at higher-than-physiological pH conditions. The fibrillar core contains residues 40-95, with both the C- and N-terminus not showing any ordered, rigid parts. The chemical shifts are similar to those recorded previously for an assigned polymorph that was fibrillized at neutral pH.

Molecular mechanisms for protein-encoded inheritance

PubMed Central

Wiltzius, Jed J. W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

2013-01-01

Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds. PMID:19684598

Sequence Variations in the Bovine Growth Hormone Gene Characterized by Single-Strand Conformation Polymorphism (Sscp) Analysis and Their Association with Milk Production Traits in Holsteins

PubMed Central

Yao, J.; Aggrey, S. E.; Zadworny, D.; Hayes, J. F.; Kuhnlein, U.

1996-01-01

Sequence variations in the bovine growth hormone (GH) gene were investigated by single strand conformation polymorphism (SSCP) analysis of seven amplified fragments covering almost the entire gene (2.7 kb). SSCPs were detected in four of these fragments and a total of six polymorphisms were found in a sample of 128 Holstein bulls. Two polymorphisms, a T->C transition in the third intron (designated GH4.1) and an A->C transversion in the fifth exon (designated GH6.2), were shown to be associated with milk production traits. GH4.1(c)/GH4.1(c) bulls had higher milk yield than GH4.1(c)/GH4.1(t) (P <= 0.005) and GH4.1(t)/GH4.1(t) (P <= 0.0022) bulls. GH4.1(c)/GH4.1(c) bulls had higher kg fat (P <= 0.0076) and protein (P <= 0.0018) than GH4.1(c)/GH4.1(t) bulls. Similar effects on milk production traits with the GH6.2 polymorphism were observed with the GH6.2(a) allele being the favorable allele. The average effects of the gene substitution for GH4.1 and GH6.2 are similar, with +/-300 kg for milk yield, +/-8 kg for fat content and +/-7 kg for protein content per lactation. The positive association of GH4.1(c) and GH6.2(a) with milk production traits may be useful for improving milk performance in dairy cattle. PMID:8978066

CD4 mimetics sensitize HIV-1-infected cells to ADCC.

PubMed

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B; Park, Jongwoo; Jones, David M; Courter, Joel R; Melillo, Bruno N; Kaufmann, Daniel E; Hahn, Beatrice H; Permar, Sallie R; Haynes, Barton F; Madani, Navid; Sodroski, Joseph G; Finzi, Andrés

2015-05-19

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

CD4 mimetics sensitize HIV-1-infected cells to ADCC

PubMed Central

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S.; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B.; Park, Jongwoo; Jones, David M.; Courter, Joel R.; Melillo, Bruno N.; Kaufmann, Daniel E.; Hahn, Beatrice H.; Permar, Sallie R.; Haynes, Barton F.; Madani, Navid; Sodroski, Joseph G.; Finzi, Andrés

2015-01-01

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection. PMID:25941367

Molecular Dynamics, Monte Carlo Simulations, and Langevin Dynamics: A Computational Review

PubMed Central

Paquet, Eric; Viktor, Herna L.

2015-01-01

Macromolecular structures, such as neuraminidases, hemagglutinins, and monoclonal antibodies, are not rigid entities. Rather, they are characterised by their flexibility, which is the result of the interaction and collective motion of their constituent atoms. This conformational diversity has a significant impact on their physicochemical and biological properties. Among these are their structural stability, the transport of ions through the M2 channel, drug resistance, macromolecular docking, binding energy, and rational epitope design. To assess these properties and to calculate the associated thermodynamical observables, the conformational space must be efficiently sampled and the dynamic of the constituent atoms must be simulated. This paper presents algorithms and techniques that address the abovementioned issues. To this end, a computational review of molecular dynamics, Monte Carlo simulations, Langevin dynamics, and free energy calculation is presented. The exposition is made from first principles to promote a better understanding of the potentialities, limitations, applications, and interrelations of these computational methods. PMID:25785262

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

PubMed Central

Kesavardhana, Sannula

2014-01-01

ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates. PMID:24920800

Active-State Model of a Dopamine D2 Receptor - Gαi Complex Stabilized by Aripiprazole-Type Partial Agonists

PubMed Central

Kling, Ralf C.; Tschammer, Nuska; Lanig, Harald; Clark, Timothy; Gmeiner, Peter

2014-01-01

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy. PMID:24932547

A low-temperature polymorph of m-quinquephenyl.

PubMed

Gomes, Ligia R; Howie, R Alan; Low, John Nicolson; Rodrigues, Ana S M C; Santos, Luís M N B F

2012-12-01

A low-temperature polymorph of 1,1':3',1'':3'',1''':3''',1''''-quinquephenyl (m-quinquephenyl), C(30)H(22), crystallizes in the space group P2(1)/c with two molecules in the asymmetric unit. The crystal is a three-component nonmerohedral twin. A previously reported room-temperature polymorph [Rabideau, Sygula, Dhar & Fronczek (1993). Chem. Commun. pp. 1795-1797] also crystallizes with two molecules in the asymmetric unit in the space group P-1. The unit-cell volume for the low-temperature polymorph is 4120.5 (4) Å(3), almost twice that of the room-temperature polymorph which is 2102.3 (6) Å(3). The molecules in both structures adopt a U-shaped conformation with similar geometric parameters. The structural packing is similar in both compounds, with the molecules lying in layers which stack perpendicular to the longest unit-cell axis. The molecules pack alternately in the layers and in the stacked columns. In both polymorphs, the only interactions between the molecules which can stabilize the packing are very weak C-H...π interactions.

Association analysis of a polymorphism of the monoamine oxidase B gene with Parkinson`s disease in a Japanese population

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

Morimoto, Yuji; Murayama, Nobuhiro; Kuwano, Akira

1995-12-18

The polymorphic allele of the monoamine oxidase B (MAO-B) gene detected by polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) was associated with Parkinson`s disease (PD) in Caucasians. We characterized this polymorphic allele, allele 1, of the MAO-B gene using direct sequencing of PCR products. A single DNA substitution (G-A), resulting gain of Mae III restriction site was detected in intron 13 of the MAO-B gene. The allele associated with PD in Caucasians was twice as frequent as in healthy Japanese, but the association of the allele of the MAO-B gene was not observed in Japanese patients with PD.more » 7 refs., 2 figs., 1 tab.« less

Polymorphism of a new Mannich base - [-4-methyl-2-((4-(4-nitrophenyl)piperazin-1-yl)methyl)phenol

NASA Astrophysics Data System (ADS)

Ayeni, Ayowole O.; Watkins, Gareth M.; Hosten, Eric C.

2018-05-01

Two polymorphs (forms I and II) of a new Mannich base 4-methyl-2-((4-(4-nitrophenyl)piperazin-1-yl)methyl)phenol have been isolated and characterized by single crystal and powder (experimental and theoretical) X-ray diffraction, thermal analysis (differential scanning calorimetry), Fourier transform infrared spectroscopy. 1H and 13C nuclear magnetic resonance spectroscopy was employed in characterising the new Mannich base. Single crystal X-ray diffraction revealed that the two polymorphs contain different conformers of the Mannich base whose hydrogen bonding schemes and packing arrangements in their respective crystals are different. Thermal analysis led to the conclusion that the two polymorphs are enantiotropically related, with a transition temperature of 138.5 °C.

Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

PubMed Central

O'Rourke, Sara M.; Schweighardt, Becky; Phung, Pham; Mesa, Kathryn A.; Vollrath, Aaron L.; Tatsuno, Gwen P.; To, Briana; Sinangil, Faruk; Limoli, Kay; Wrin, Terri

2012-01-01

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals. PMID:22933284

G-Quadruplex conformational change driven by pH variation with potential application as a nanoswitch.

PubMed

Yan, Yi-Yong; Tan, Jia-Heng; Lu, Yu-Jing; Yan, Siu-Cheong; Wong, Kwok-Yin; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

2013-10-01

G-Quadruplex is a highly polymorphic structure, and its behavior in acidic condition has not been well studied. Circular dichroism (CD) spectra were used to study the conformational change of G-quadruplex. The thermal stabilities of the G-quadruplex were measured with CD melting. Interconversion kinetics profiles were investigated by using CD kinetics. The fluorescence of the inserted 2-Aminopurine (Ap) was monitored during pH change and acrylamide quenching, indicating the status of the loop. Proton NMR was adopted to help illustrate the change of the conformation. G-Quadruplex of specific loop was found to be able to transform upon pH variation. The transformation was resulted from the loop rearrangement. After screening of a library of diverse G-quadruplex, a sequence exhibiting the best transformation property was found. A pH-driven nanoswitch with three gears was obtained based on this transition cycle. Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. It can be used to design pH-driven nanodevices such as a nanoswitch. These results provide more insights into G-quadruplex polymorphism, and also contribute to the design of DNA-based nanomachines and logic gates. © 2013.

Epitope mapping of tsh receptor-blocking antibodies in Graves' disease that appear during pregnancy.

PubMed

Kung, A W; Lau, K S; Kohn, L D

2001-08-01

Spontaneous remission of Graves' disease during pregnancy is thought to be due to a reduction of thyroid-stimulating antibody activity. We suspected, however, that a broader change in TSH receptor antibody characteristics might play an important role in modulating disease activity during pregnancy. We measured TSH binding inhibitory Ig, thyroid-stimulating antibody, and thyroid stimulating-blocking antibody activities in 13 pregnant Graves' disease patients at first, second, and third trimesters and 4 months postpartum. To measure and epitope-map thyroid-stimulating antibody and thyroid stimulating-blocking antibody activities, we used CHO cells transfected with wild-type human TSH receptor or with several TSH receptor-LH/hCG receptor chimeras: Mc1+2, Mc2, and Mc4. These chimeric cells have their respective TSH receptor residues 9-165, 90-165, and 261-370 substituted with equivalent residues of the LH/hCG receptor. Overall thyroid-stimulating antibody decreased, whereas thyroid stimulating-blocking antibody increased progressively during pregnancy. TSH binding inhibitory Ig fluctuated in individual patients, but overall the activities remained statistically unchanged. Thyroid stimulating-blocking antibody appeared in subjects who were either negative for thyroid-stimulating antibody or whose thyroid-stimulating antibody activity increased or decreased during pregnancy. Epitope mapping showed that the thyroid-stimulating antibodies were mainly directed against residues 9-165 of the N-terminus of the TSH receptor extracellular domain. All thyroid stimulating-blocking antibodies had blocking activities against residues 261-370 of the C-terminus of the ectodomain. However, the majority of the thyroid stimulating-blocking antibodies had a hybrid conformational epitope directed against N-terminal residues 9-89 or 90-165 as well. Despite a change in the activity level, we did not observe any change in the epitope of either the stimulatory or blocking Abs as pregnancy advanced. In conclusion, a change in the specificity of TSH receptor antibody from stimulatory to blocking activity was observed during pregnancy, and the appearance of thyroid stimulating-blocking antibody may contribute to the remission of Graves' disease during pregnancy.

Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

PubMed

Hu, A; Norrby, E

1994-09-01

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.

Antigenic Variation of East/Central/South African and Asian Chikungunya Virus Genotypes in Neutralization by Immune Sera

PubMed Central

Chua, Chong-Long; Sam, I-Ching; Merits, Andres; Chan, Yoke-Fun

2016-01-01

Background Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood. Methodology/Principal Findings We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008–2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes. Conclusion/Significance Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays. PMID:27571254

Gene analysis of steroid 5 alpha-reductase 1 in hyperandrogenic women.

PubMed

Eminović, Izet; Komel, Radovan; Prezelj, Janez; Karamehić, Jasenko; Gavrankapetanović, Faris; Heljić, Becir

2005-08-01

To examine the gene encoding for 5alpha-reductase type 1 in hyperandrogenic women, and assess the association of its eventual mutations or polymorphisms with the development of the hyperandrogenic female pattern. Sixteen hyperandrogenic women were included in the study. Single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing were performed after polymerase chain reaction amplification of each of the 5 exons of the SRD5A1 gene in both hyperandrogenic and control group (16 participants). Sequence analysis identified the existence of many polymorphisms; in codon 24 of exon 1, GGC (Gly) into GAC (Asp); in codon 30 of exon 1, CGG (Arg) into CGC (Arg); in exon 3 codon 169, ACA to ACG (both encoding for threonine); in exon 5, AGA to AGG (both encoding for arginine, codon 260); and T/C polymorphism in intron 2. Polymorphisms were found in both groups. Polymorphisms of SRD5A1 gene were the same in both hyperandrogenic and healthy women, indicating no significant associations of genetic polymorphisms/variations of SRD5A1 gene with clinical manifestations of hyperandrogenic disorders in women.

Structural analyses of polymorphic transitions of sn-1, 3-distearoyl-2-oleoylglycerol (SOS) and sn-1, 3-dioleoyl-2-stearoylglycerol (OSO): assessment on steric hindrance of unsaturated and saturated acyl chain interactions.

PubMed

Yano, J; Sato, K; Kaneko, F; Small, D M; Kodali, D R

1999-01-01

Polymorphic transformations in two saturated-unsaturated mixed acid triacylglycerols, SOS (sn -1,3-distearoyl-2-oleoylglycerol) and OSO (sn -1,3-dioleoyl-2-stearoylglycerol), have been studied by FT-IR spectroscopy using deuterated specimens in which stearoyl chains are fully deuterated. A reversible phase transition between sub alpha and alpha and a series of irreversible transitions (alpha-->gamma-->beta'-->beta (beta2, beta1) for SOS and alpha-->beta'-->beta for OSO) were studied with an emphasis on the conformational ordering process of stearoyl and oleoyl chains. The alpha-->sub alpha reversible transition was due to the orientational change of stearoyl chains in the lateral directions from the hexagonal subcell to a perpendicularly packed one. As the first stage of the series of irreversible transitions from alpha to beta, the conformational ordering of saturated chains took place in the alpha-->gamma transition of SOS and in the alpha-->beta' transition of OSO; one stearoyl chain in SOS and OSO takes the all-trans conformation and the second stearoyl chain in SOS takes the bent conformation like those observed in the most stable beta-type. As the final stage, the ordering of unsaturated chains occurred in the beta'-->beta transition both for SOS and OSO. A conversion in the layered structure from bilayer to trilayer was also accompanied by the conformational ordering in the alpha-->gamma transition of SOS and in the beta'-->beta transition of OSO.

Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer's disease.

PubMed

Rasmussen, Jay; Mahler, Jasmin; Beschorner, Natalie; Kaeser, Stephan A; Häsler, Lisa M; Baumann, Frank; Nyström, Sofie; Portelius, Erik; Blennow, Kaj; Lashley, Tammaryn; Fox, Nick C; Sepulveda-Falla, Diego; Glatzel, Markus; Oblak, Adrian L; Ghetti, Bernardino; Nilsson, K Peter R; Hammarström, Per; Staufenbiel, Matthias; Walker, Lary C; Jucker, Mathias

2017-12-05

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype.

Structure-Based Design of a Soluble Prefusion-Closed HIV-1 Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity

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

Chuang, Gwo-Yu; Geng, Hui; Pancera, Marie

ABSTRACT The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, andmore » colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties. IMPORTANCEOne approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.« less

Role of tolerogen conformation in induction of oral tolerance in experimental autoimmune myasthenia gravis.

PubMed

Im, S H; Barchan, D; Souroujon, M C; Fuchs, S

2000-10-01

We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.

Polymorphism of DNA conformation inside the bacteriophage capsid.

PubMed

Leforestier, Amélie

2013-03-01

Double-stranded DNA bacteriophage genomes are packaged into their icosahedral capsids at the highest densities known so far (about 50 % w:v). How the molecule is folded at such density and how its conformation changes upon ejection or packaging are fascinating questions still largely open. We review cryo-TEM analyses of DNA conformation inside partially filled capsids as a function of the physico-chemical environment (ions, osmotic pressure, temperature). We show that there exists a wide variety of DNA conformations. Strikingly, the different observed structures can be described by some of the different models proposed over the years for DNA organisation inside bacteriophage capsids: either spool-like structures with axial or concentric symmetries, or liquid crystalline structures characterised by a DNA homogeneous density. The relevance of these conformations for the understanding of DNA folding and unfolding upon ejection and packaging in vivo is discussed.

Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

PubMed Central

Fong, Rachel H.; Banik, Soma S. R.; Mattia, Kimberly; Barnes, Trevor; Tucker, David; Liss, Nathan; Lu, Kai; Selvarajah, Suganya; Srinivasan, Surabhi; Mabila, Manu; Miller, Adam; Muench, Marcus O.; Michault, Alain; Rucker, Joseph B.; Paes, Cheryl; Simmons, Graham; Kahle, Kristen M.

2014-01-01

ABSTRACT Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response. PMID:25275138

ACE phenotyping in human heart

PubMed Central

Tikhomirova, Victoria E.; Kost, Olga A.; Kryukova, Olga V.; Golukhova, Elena Z.; Bulaeva, Naida I.; Zholbaeva, Aigerim Z.; Bokeria, Leo A.; Garcia, Joe G. N.

2017-01-01

Aims Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs. Methods and results We performed ACE phenotyping (ACE levels, conformation and kinetic characteristics) in the human heart and compared it with that in the lung. ACE activity in heart tissues was 10–15 lower than that in lung. Various ACE effectors, LMW endogenous ACE inhibitors and HMW ACE-binding partners, were shown to be present in both heart and lung tissues. “Conformational fingerprint” of heart ACE (i.e., the pattern of 17 mAbs binding to different epitopes on the ACE surface) significantly differed from that of lung ACE, which reflects differences in the local conformations of these ACEs, likely controlled by different ACE glycosylation in these organs. Substrate specificity and pH-optima of the heart and lung ACEs also differed. Moreover, even within heart the apparent ACE activities, the local ACE conformations, and the content of ACE inhibitors differ in atria and ventricles. Conclusions Significant differences in the local conformations and kinetic properties of heart and lung ACEs demonstrate tissue specificity of ACE and provide a structural base for the development of mAbs able to distinguish heart and lung ACEs as a potential blood test for predicting atrial fibrillation risk. PMID:28771512

TRALI ASSOCIATED HNA-3a ANTIBODIES RECOGNIZE COMPLEX DETERMINANTS ON CHOLINE TRANSPORTER-LIKE PROTEIN 2 (CTL2)

PubMed Central

Bougie, Daniel W; Peterson, Julie A; Kanack, Adam J; Curtis, Brian R; Aster, Richard H

2014-01-01

Background HNA-3a specific antibodies can cause severe, sometimes fatal, transfusion related acute lung injury (TRALI) when present in transfused blood. The HNA3-a/b antigens are determined by an R154Q polymorphism in the first of five extracellular loops of the 10-membrane spanning choline transporter-like protein 2 (CTL2) expressed on neutrophils, lymphocytes and other tissues. About 50% of HNA-3a antibodies (Type 1) can be detected using CTL2 Loop 1 peptides containing R154; the remaining 50% (Type 2) fail to recognize this target. Understanding the basis for this difference could guide efforts to develop practical assays to screen blood donors for HNA-3 antibodies. Study design and methods Reactions of HNA-3a antibodies against recombinant versions of human, mouse, and human/mouse (chimeric) CTL2 were characterized using flow cytometry and various solid phase assays. Results Findings made show that, for binding to CTL2, Type 2 HNA-3a antibodies require non-polymorphic amino acid residues in the third, and possibly the second, extracellular loops of CTL2 to be in a configuration comparable to that found naturally in the cell membrane. In contrast, Type 1 antibodies require only peptides from the first extracellular loop that contain R154 for recognition. Conclusion Although Type 1 HNA-3a antibodies can readily be detected in solid phase assays that use a CTL2 peptide containing R154 as a target, development of a practical test to screen blood donors for Type 2 antibodies will pose a serious technical challenge because of the complex nature of the epitope(s) recognized by this antibody sub-group. PMID:24846273

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

PubMed Central

2010-01-01

Background Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. Methods To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. Results The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion. PMID:21092207

Structural Basis for FcγRIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

PubMed Central

Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.; Tan Sardjono, Caroline; Esparon, Sandra; Trist, Halina M.; Powell, Maree S.; Szee Tan, Peck; Cendron, Angela C.; Wines, Bruce D.; Scott, Andrew M.; Hogarth, P. Mark

2012-01-01

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag–Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly. PMID:21856937

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.

CE-BLAST makes it possible to compute antigenic similarity for newly emerging pathogens.

PubMed

Qiu, Tianyi; Yang, Yiyan; Qiu, Jingxuan; Huang, Yang; Xu, Tianlei; Xiao, Han; Wu, Dingfeng; Zhang, Qingchen; Zhou, Chen; Zhang, Xiaoyan; Tang, Kailin; Xu, Jianqing; Cao, Zhiwei

2018-05-02

Major challenges in vaccine development include rapidly selecting or designing immunogens for raising cross-protective immunity against different intra- or inter-subtypic pathogens, especially for the newly emerging varieties. Here we propose a computational method, Conformational Epitope (CE)-BLAST, for calculating the antigenic similarity among different pathogens with stable and high performance, which is independent of the prior binding-assay information, unlike the currently available models that heavily rely on the historical experimental data. Tool validation incorporates influenza-related experimental data sufficient for stability and reliability determination. Application to dengue-related data demonstrates high harmonization between the computed clusters and the experimental serological data, undetectable by classical grouping. CE-BLAST identifies the potential cross-reactive epitope between the recent zika pathogen and the dengue virus, precisely corroborated by experimental data. The high performance of the pathogens without the experimental binding data suggests the potential utility of CE-BLAST to rapidly design cross-protective vaccines or promptly determine the efficacy of the currently marketed vaccine against emerging pathogens, which are the critical factors for containing emerging disease outbreaks.

Immunoreactivity of Biochemically Purified Amandin from Thermally Processed Almonds (Prunus dulcis L.).

PubMed

Zaffran, Valerie D; Sathe, Shridhar K

2018-06-15

Almond seeds were subjected to select thermal processing and amandin was purified from processed and unprocessed (control) seeds using cryoprecipitation. Amandin immunoreactivity was assessed using two murine monoclonal antibodies (mAbs)-4C10 and 4F10 detecting human IgE-relevant conformational and linear epitopes, respectively. Overall amandin immunoreactivity following thermal treatment ranged from 64.9% to 277.8% (4C10) and 81.3% to 270.3% (4F10). Except for autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min), the tested processing conditions resulted in increased immunoreactivity as determined by mAbs 4C10 and 4F10-based enzyme-linked immunosorbent assays (ELISAs). A significant, yet not complete, reduction in immunoreactivity was caused by autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min). Western- and dot-blot immunoassays corroborated the ELISA results, confirming amandin thermal stability. The tested immunoassays indicated amandin to be stable, regardless of the targeted epitope and the processing method that whole almond seeds were subjected to. © 2018 Institute of Food Technologists®.

Specificity of Mimotope-Induced Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Antibodies Does Not Ensure Biological Activity

PubMed Central

Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-01-01

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes. PMID:21573118

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity.

PubMed

Latzka, Julia; Gaier, Sonja; Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-05-06

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections

PubMed Central

Kam, Yiu-Wing; Lee, Cheryl Yi-Pin; Teo, Teck-Hui; Howland, Shanshan W.; Amrun, Siti Naqiah; See, Peter; Kng, Nicholas Qing-Rong; Huber, Roland G.; Xu, Mei-Hui; Tan, Heng-Liang; Choo, Andre; Ginhoux, Florent; Fink, Katja; Wang, Cheng-I; Ng, Lisa F.P.

2017-01-01

Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitopes on the envelope (E) glycoprotein: the highly conserved fusion-loop peptide, a conformation-specific epitope on the E monomer, and a quaternary epitope on the virion surface. The most potent ZIKV-neutralizing mAb (SIgN-3C) was assessed in 2 type I interferon receptor–deficient (IFNAR–/–) mouse models of ZIKV infection. Treatment of adult nonpregnant mice with SIgN-3C rescued mice from virus-induced weight loss and mortality. The SIgN-3C variant with Leu-to-Ala mutations in the Fc region (SIgN-3C-LALA) did not induce antibody-dependent enhancement (ADE) in vitro but provided similar levels of protection in vivo. In pregnant ZIKV-infected IFNAR–/– mice, treatment with SIgN-3C or SIgN-3C-LALA significantly reduced viral load in the fetal organs and placenta and abrogated virus-induced fetal growth retardation. Therefore, SIgN-3C-LALA holds promise as a ZIKV prophylactic and therapeutic agent. PMID:28422757

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

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

Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.

2015-09-15

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope.more » Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.« less

Impact of clonal competition for peptide-MHC complexes on the CD8[superscript +] T-cell repertoire selection in a persistent viral infection

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

Wynn, Katherine K.; Fulton, Zara; Cooper, Leanne

2008-04-29

CD8{sup +} T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)-specific CD8{sup +} T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident withmore » an atypical major histocompatibility complex (MHC)-peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more 'featureless' landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.« less

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

Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution.

PubMed

Marez, D; Legrand, M; Sabbagh, N; Lo Guidice, J M; Spire, C; Lafitte, J J; Meyer, U A; Broly, F

1997-06-01

The polymorphic cytochrome P450 CYP2D6 is involved in the metabolism of various drugs of wide therapeutic use and is a presumed susceptibility factor for certain environmentally-induced diseases. Our aim was to define the mutations and alleles of the CYP2D6 gene and to evaluate their frequencies in the European population. Using polymerase chain reaction-single strand conformation polymorphism analysis, 672 unrelated subjects were screened for mutations in the 9 exons of the gene and their exon-intron boundaries. A total of 48 point mutations were identified, of which 29 were novel. Mutations 1749 G-->C, 2938 C-->T and 4268 G-->C represented 52.6%, 34.3% and 52.9% of the mutations in the total population, respectively. Of the eight detrimental mutations detected, the 1934 G-->A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *2, *2B, *4A and *5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.

Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

PubMed

Tashakori, Mahnaz; Mahnaz, Tashakori; Kuhls, Katrin; Katrin, Kuhls; Al-Jawabreh, Amer; Amer, Al-Jawabreh; Mauricio, Isabel L; Isabel, Mauricio; Schönian, Gabriele; Gabriele, Schönian; Farajnia, Safar; Safar, Farajnia; Alimohammadian, Mohammad Hossein; Hossein, Alimohammadian Mohammad

2006-04-01

Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

X-ray structural studies and physicochemical characterization of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl- 3,4-dihydro-2(1H)-pyrimidinone polymorphs.

PubMed

Miyamae, A; Kitamura, S; Tada, T; Koda, S; Yasuda, T

1991-10-01

The polymorphism of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-di hydro- 2(1 H)-pyrimidinone (FK664; 1) was characterized by using X-ray powder diffractometry, differential scanning calorimetry (DSC), and IR spectroscopy. Structures of two polymorphs (Forms A and B) were determined by X-ray crystallographic analysis. Form A crystallized in the monoclinic space group P2(1)/c, with a = 13.504(2), b = 6.733(1), c = 24.910(8) A, beta = 96.55(4) degrees, z = 4, and dcal = 1.203 g/cm3, while Form B crystallized in the same space group, with a = 8.067(2), b = 15.128(4), c = 18.657(4) A, beta = 102.34(3) degrees, z = 4, and dcal = 1.216 g/cm3. The conformational features of 1 were very similar between the two polymorphs. Compound 1, in both crystal forms, took an energetically reasonable conformation in three rigid planes, such as 2-pyrimidone, trimethylphenyl, and dimethoxyphenyl rings, but the molecules were packed in different ways between the two polymorphs. In the Form B crystal, a short contact was possible, to form pi-pi interactions between two dimethoxyphenyl groups related with the inversion center in the crystal lattice; this interaction seems to contribute to stabilizing the crystal structure of Form B. Both Forms A and B showed only one endothermic peak due to fusion at 115 and 140 degrees C, respectively, on the DSC thermograms; therefore, it is suggested that there are no transition points between the two polymorphs. The heats of fusion obtained from the DSC thermograms were 33.2(2) kJ/mol for Form A and 36.8(1) kJ/mol for Form B.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

PubMed Central

Neudecker, Philipp; Lehmann, Katrin; Nerkamp, Jörg; Haase, Tanja; Wangorsch, Andrea; Fötisch, Kay; Hoffmann, Silke; Rösch, Paul; Vieths, Stefan; Scheurer, Stephan

2003-01-01

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy. PMID:12943529

HLA-DM mediates epitope selection by a "compare-exchange" mechanism when a potential peptide pool is available.

PubMed

Ferrante, Andrea; Anderson, Matthew W; Klug, Candice S; Gorski, Jack

2008-01-01

HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a "compare-exchange" sorting algorithm on an available peptide pool. Such a "third party"-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

PubMed

Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

1994-12-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

EpHLA: an innovative and user-friendly software automating the HLAMatchmaker algorithm for antibody analysis.

PubMed

Sousa, Luiz Cláudio Demes da Mata; Filho, Herton Luiz Alves Sales; Von Glehn, Cristina de Queiroz Carrascosa; da Silva, Adalberto Socorro; Neto, Pedro de Alcântara dos Santos; de Castro, José Adail Fonseca; do Monte, Semíramis Jamil Hadad

2011-12-01

The global challenge for solid organ transplantation programs is to distribute organs to the highly sensitized recipients. The purpose of this work is to describe and test the functionality of the EpHLA software, a program that automates the analysis of acceptable and unacceptable HLA epitopes on the basis of the HLAMatchmaker algorithm. HLAMatchmaker considers small configurations of polymorphic residues referred to as eplets as essential components of HLA-epitopes. Currently, the analyses require the creation of temporary files and the manual cut and paste of laboratory tests results between electronic spreadsheets, which is time-consuming and prone to administrative errors. The EpHLA software was developed in Object Pascal programming language and uses the HLAMatchmaker algorithm to generate histocompatibility reports. The automated generation of reports requires the integration of files containing the results of laboratory tests (HLA typing, anti-HLA antibody signature) and public data banks (NMDP, IMGT). The integration and the access to this data were accomplished by means of the framework called eDAFramework. The eDAFramework was developed in Object Pascal and PHP and it provides data access functionalities for software developed in these languages. The tool functionality was successfully tested in comparison to actual, manually derived reports of patients from a renal transplantation program with related donors. We successfully developed software, which enables the automated definition of the epitope specificities of HLA antibodies. This new tool will benefit the management of recipient/donor pairs selection for highly sensitized patients. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Rodríguez-Romero, Adela, E-mail: adela@unam.mx; Hernández-Santoyo, Alejandra; Fuentes-Silva, Deyanira

This study describes the three-dimensional structure of the endogenous glycosylated allergen Hev b 2 (endo-β-1,3-glucanase), which exhibits three post-translational modifications that form a patch on the surface of the molecule that is proposed to be an allergenic IgE epitope. Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibitsmore » three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.« less

A novel single-nucleotide polymorphism of the visfatin gene and its associations with performance traits in the chicken.

PubMed

Han, R-L; Lan, X-Y; Zhang, L-Z; Ren, G; Jing, Y-J; Li, M-J; Zhang, B; Zhao, M; Guo, Y-K; Kang, X-T; Chen, H

2010-01-01

Visfatin is a peptide that is predominantly expressed in visceral adipose tissue and is hypothesized to be related to obesity and insulin resistance. In this study, a novel silent single-nucleotide polymorphism (SNP) was found in exon 7 of the chicken visfatin gene (also known as PBEF1) by single-stranded conformation polymorphism (SSCP) and DNA sequencing. In total, 836 chickens forming an F2 resource population of Gushi chicken crossed with Anka broiler were genotyped by XbaI forced RFLP, and the associations of this polymorphism with chicken growth, carcass characteristics, and meat quality were analyzed. Significant associations were found between the polymorphism and 4-week body weight (BW4), 6-week body weight (BW6), 4-week body slanting length (BSL4), fat bandwidth (FBW), breast muscle water loss rate (BWLR) and breast muscle fiber density (BFD) (P < 0.05), as well as 4-week breastbone length (BBL4) (P < 0.01). These observations suggested that the polymorphism in exon7 of the visfatin gene had significant effects on the early growth traits of chicken.

[Association analysis between SNPs of the growth hormone receptor gene and growth traits in arctic fox].

PubMed

DU, Zhi-Heng; Liu, Zong-Yue; Bai, Xiu-Juan

2010-06-01

Using single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing, single nucleotide polymorphisms (SNPs) of growth hormone receptor (GHR) gene were detected in an arctic fox population. Correlation analysis between GHR polymorphisms and growth traits were carried out using the appropriate model. Four SNPs, G3A in the 5'UTR, C99T in the first exon, T59C and G65A in the fifth exon were identified on the arctic fox GHR gene. The G3A and C99T polymorphisms of GHR were associated with female fox body weight (Pamp;0.05) and the T59C and G65A polymorphisms of GHR were associated with male fox body weight (Pamp;0.05) and the skin length of the female fox (Pamp;0.01). Therefore, marker assistant selection on body weight and skin length of arctic foxes using these SNPs can be applied to get big and high quality arctic foxes.

Allele frequency and genotype distribution of polymorphisms within disease-related genes is influenced by ethnic population sub-structuring in Sudan.

PubMed

Bereir, R E H; Mohamed, H S; Seielstad, M; El Hassani, A M; Khalil, E A G; Peacock, C S; Blackwell, J M; Ibrahim, M E

2003-09-01

Four single nucleotide polymorphisms (SNPs) and a variable number of tandem repeats (VNTR) polymorphism located within disease associated/causing genes were typed in four populations of different tribal and ethnic affiliation from the Sudan. The genotype and allele frequencies were compared with those of other groups from published and unpublished data of world populations. The combined Sudanese sample conformed with Hardy-Weinberg equilibrium (HWE) expectation. However, population sub-structuring according to ethnic/linguistic group indicated at least two SNPs in departure from HWE. Differences in allele frequencies and genotype distribution between groups was also noted in three of the four SNPs. The other loci were distributed homogeneously within the populations studied with genotype frequencies in agreement with HWE expectation. These results highlight the importance of inter-population stratification for polymorphic markers, as well as the potential influence of evolutionary history and ethnic variation of loci, in the general distribution of SNPs and other polymorphisms.

Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity

PubMed Central

Luo, Jie; Taylor, Palmer; Losen, Mario; de Baets, Marc H.; Shelton, G. Diane; Lindstrom, Jon

2009-01-01

The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of α1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human α1 subunits with α7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop α1 67–76 in combination with the N-terminal α helix α1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to α1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same α1 MIR sequences substituted was recognized by a majority of human, feline, and canine MG sera. The presence of the α1 MIR sequences in α1/α7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR. PMID:19890000

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection.

PubMed

Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Marceau, Gabriel; Beland, Kathie; Alvarez, Fernando

2004-05-01

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.

Comprehensive 3D-modeling of allergenic proteins and amino acid composition of potential conformational IgE epitopes

PubMed Central

Oezguen, Numan; Zhou, Bin; Negi, Surendra S.; Ivanciuc, Ovidiu; Schein, Catherine H.; Labesse, Gilles; Braun, Werner

2008-01-01

Similarities in sequences and 3D structures of allergenic proteins provide vital clues to identify clinically relevant IgE cross-reactivities. However, experimental 3D structures are available in the Protein Data Bank for only 5% (45/829) of all allergens catalogued in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP). Here, an automated procedure was used to prepare 3D-models of all allergens where there was no experimentally determined 3D structure or high identity (95%) to another protein of known 3D structure. After a final selection by quality criteria, 433 reliable 3D models were retained and are available from our SDAP Website. The new 3D models extensively enhance our knowledge of allergen structures. As an example of their use, experimentally derived “continuous IgE epitopes” were mapped on 3 experimentally determined structures and 13 of our 3D-models of allergenic proteins. Large portions of these continuous sequences are not entirely on the surface and therefore cannot interact with IgE or other proteins. Only the surface exposed residues are constituents of “conformational IgE epitopes” which are not in all cases continuous in sequence. The surface exposed parts of the experimental determined continuous IgE epitopes showed a distinct statistical distribution as compared to their presence in typical protein-protein interfaces. The amino acids Ala, Ser, Asn, Gly and particularly Lys have a high propensity to occur in IgE binding sites. The 3D-models will facilitate further analysis of the common properties of IgE binding sites of allergenic proteins. PMID:18621419

Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

PubMed

Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

2017-05-01

Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

PubMed

Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

2010-11-01

As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

The tryptic cleavage product of the mature form of the bovine desmoglein 1 ectodomain is one of the antigen moieties immunoprecipitated by all sera from symptomatic patients affected by a new variant of endemic pemphigus.

PubMed

Abréu-Vélez, Ana María; Javier Patiño, Pablo; Montoya, Fernando; Bollag, Wendy B

2003-01-01

Multiple antigens are recognized by sera from patients with pemphigus foliaceus (PF). Several have been identified including keratin 59, desmocollins, envoplakin, periplakin, and desmogleins 1 and 3 (Dsg1 and Dsg3). In addition, an 80 kDa antigen was identified as the N-terminal fragment of Dsg1 using as antigen source an insoluble epidermal cell envelope preparation. However, still unsolved was the identity of the most important antigenic moiety, a 45 kDa tryptic fragment which is recognized by all sera from patients with fogo selvagem, pemphigus foliaceus, by half of pemphigus vulgaris sera and by a new variant of endemic pemphigus in E1 Bagre, Colombia that resembles Senear-Usher syndrome. Here, we report the identification of the 45 kDa conformational epitope of a soluble tryptic cleavage product from viable bovine epidermis. To elucidate the nature of this peptide, viable bovine epidermis was trypsin-digested, and glycosylated peptides were partially purified on a concanavalin A (Con-A) affinity column. This column fraction was then used as an antigen source for further immunoaffinity purification. A PF patient's serum covalently coupled to a Staphylococcus aureus protein A column was incubated with the Con-A eluted products and the immuno-isolated antigen was separated by SDS-PAGE, transferred to a membrane, and visualized with Coomassie blue, silver and amido black stains. The 45 kD band was subjected to amino acid sequence analysis revealing the sequence, EXIKFAAAXREGED, which matched the mature form of the extracellular domain of bovine Dsg1. This study confirms the biological importance of the ectodomain of Dsg1 as well as the relevance of conformational epitopes in various types of pemphigus.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

PubMed

Motamedi-Shad, Neda; Jagger, Alistair M; Liedtke, Maximilian; Faull, Sarah V; Nanda, Arjun Scott; Salvadori, Enrico; Wort, Joshua L; Kay, Christopher W M; Heyer-Chauhan, Narinder; Miranda, Elena; Perez, Juan; Ordóñez, Adriana; Haq, Imran; Irving, James A; Lomas, David A

2016-10-01

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation. © 2016 The Author(s).

Polymorphism in and localization of the gene LCP2 (SLP-76) to chromosome 5q33.1-qter

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

Sunden, S.L.F.; Carr, L.L.; Clements, J.L.

This report describes the localization of the human LCP2 gene to human chromosome 5q33.1-qter using single-stranded conformation polymorphisms analysis. This gene encodes an SH2 domain containing leukocyte protein of 76 kDa (SLP-76), which plays a functional role in T-cell activation. It remains to be determined whether mutations in this gene or translocations at this chromosome location are the genetic basis for various diseases, including lymphoblastic leukemia. 12 refs., 1 fig.

Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human beta-globin gene cluster.

PubMed

Kaushik, Mahima; Kukreti, Shrikant

2006-01-01

Structural polymorphism of DNA is a widely accepted property. A simple addition to this perception has been our recent finding, where a single nucleotide polymorphism (SNP) site present in a quasipalindromic sequence of beta-globin LCR exhibited a hairpin-duplex equilibrium. Our current studies explore that secondary structures adopted by individual complementary strands compete with formation of a perfect duplex. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we have demonstrated the structural transitions within a perfect duplex containing 11 bp quasipalindromic stretch (TGGGG(G/C)CCCCA), to hairpins and bulge duplex forms. The extended version of the 11 bp duplex, flanked by 5 bp on both sides also demonstrated conformational equilibrium between duplex and hairpin species. Gel-electrophoresis confirms that the duplex coexists with hairpin and bulge duplex/cruciform species. Further, in CD spectra of duplexes, presence of two overlapping positive peaks at 265 and 285 nm suggest the features of A- as well as B-type DNA conformation and show oligomer concentration dependence, manifested in A --> B transition. This indicates the possibility of an architectural switching at quasipalindromic region between linear duplex to a cruciform structure. Such DNA structural variations are likely to be found in the mechanics of molecular recognition and manipulation by proteins.

Vibrational spectroscopic study on polymorphism of erucic acid and palmitoleic acid: γ1→α1 and γ→α reversible solid state phase transitions

NASA Astrophysics Data System (ADS)

Kaneko, Fumitoshi; Yamazaki, Kazuhiro; Kobayashi, Masamichi; Sato, Kiyotaka; Suzuki, Masao

1994-08-01

The infrared and Raman spectra of four polymorphic phases (α, α1, γ and γ1) of erucic acid ( cis-13-docosenoic acid) and those of two polymorphic phases (α and γ) of palmitoleic acid ( cis-9-hexadecenoic acid) were investigated. The γ and γ1 phases of erucic acid were analyzed on the basis of crystal structures determined by us. There were large spectral differences between γ and γ1 phases, which could be ascribed to the differences in the conformation of cis-olefin groups and the subcell structure. Two types of reversible solid state phase transitions (γ→α and γ1→α1 transitions) were followed by the infrared and Raman spectra. It was concluded that the mechanism of the γ→α phase transition of erucic and palmitoleic acids is essentially the same as that of oleic acid previously reported by us [ J. Phys. Chem.90, 6371 (1986)], i.e. this phase transition is of order-disorder type accompanied by a conformational disordering at the methyl-terminal chain. Spectral changes on the γ1→α1 transition suggested that a similar structural change took place during this transition but there were large structural differences between α and α1.

PGV04, an HIV-1 gp120 CD4 Binding Site Antibody, Is Broad and Potent in Neutralization but Does Not Induce Conformational Changes Characteristic of CD4

PubMed Central

Falkowska, Emilia; Ramos, Alejandra; Feng, Yu; Zhou, Tongqing; Moquin, Stephanie; Walker, Laura M.; Wu, Xueling; Seaman, Michael S.; Wrin, Terri; Kwong, Peter D.; Wyatt, Richard T.; Mascola, John R.; Poignard, Pascal

2012-01-01

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1. PMID:22345481

Ionic tethering contributes to the conformational stability and function of complement C3b.

PubMed

López-Perrote, Andrés; Harrison, Reed E S; Subías, Marta; Alcorlo, Martín; Rodríguez de Córdoba, Santiago; Morikis, Dimitrios; Llorca, Oscar

2017-05-01

C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular evolution of the leptin exon 3 in some species of the family Canidae.

PubMed

Chmurzynska, Agata; Zajac, Magdalena; Switonski, Marek

2003-01-01

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris)--16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.

Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1.

PubMed

Stamogiannos, Athanasios; Maben, Zachary; Papakyriakou, Athanasios; Mpakali, Anastasia; Kokkala, Paraskevi; Georgiadis, Dimitris; Stern, Lawrence J; Stratikos, Efstratios

2017-03-14

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

Synthesis, characterization, crystal structure and DFT study of two new polymorphs of a Schiff base (E)-2-((2,6-dichlorobenzylidene)amino)benzonitrile

NASA Astrophysics Data System (ADS)

Benarous, N.; Cherouana, A.; Aubert, Emmanuel; Durand, Pierrick; Dahaoui, S.

2016-02-01

Two new polymorphs of Schiff base, (E)-2-((2,6-dichlorobenzylidene)amino)benzonitrile, were prepared from the condensation of 4-amino-benzonitrile and 2,6-dichlorobenzaldehyde. The two polymorphs crystallize in two different space groups: P21/c for polymorph (I) with volume 1264.23(2) Å3 and Pbca for polymorph (II) with volume 2469.3(2) Å3. The two polymorphs have been characterized by FT-IR and UV-VIS spectroscopy. The crystal structures of both compounds were determined by single X-ray analysis. The difference between the two polymorphs was observed at the angle between the two phenyl rings which is 4.81° for the first one and 82.27° for the second one. Both crystal structures are built on the basis of moderate and weak hydrogen bonds. Theoretical calculations on isolated molecules at the MP2 cc-pVDZ level show that the two polymorphs correspond to two molecular conformations that are within less than 1 kJ mol-1 and DFT periodic calculations indicate that (II) is more stable than (I) by 4.1 kJ mol-1 of formula unit. Additionally, we performed TD-DFT calculation for free ligands to support the experimental data.

Discovery of Cellulose Surface Layer Conformation by Nonlinear Vibrational Spectroscopy

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

Zhang, Libing; Fu, Li; Wang, Hong-fei

2017-03-14

Significant questions remain with respect to the structure and polymorphs of cellulose. These include the cellulose surface layers and the bulk crystalline core as well as the conformational differences. The Total Internal Reflection Sum Frequency Generation Vibrational Spectroscopy (TIR-SFG-VS) combined with the conventional SFG-VS (non-TIR) can help to resolve these questions by selectively characterizing the molecular structures of surface layers and the crystalline core of cellulose. From the SFG spectra in the C-H and O-H regions, we found that the surface layers of Avicel are essentially amorphous; while the surface layers of Iβ cellulose are crystalline but with different structuralmore » and spectroscopic signatures than that of its crystalline core. This work demonstrates the capacity of TIR and Non-TIR SFG-VS tools in selectively studying the structures and polymorphs of cellulose. In addition, these results also suggest that the assignments of major vibrational peaks for cellulose need to be further determined.« less

Genetic polymorphism and natural selection of Duffy binding protein of Plasmodium vivax Myanmar isolates

PubMed Central

2012-01-01

Background Plasmodium vivax Duffy binding protein (PvDBP) plays an essential role in erythrocyte invasion and a potential asexual blood stage vaccine candidate antigen against P. vivax. The polymorphic nature of PvDBP, particularly amino terminal cysteine-rich region (PvDBPII), represents a major impediment to the successful design of a protective vaccine against vivax malaria. In this study, the genetic polymorphism and natural selection at PvDBPII among Myanmar P. vivax isolates were analysed. Methods Fifty-four P. vivax infected blood samples collected from patients in Myanmar were used. The region flanking PvDBPII was amplified by PCR, cloned into Escherichia coli, and sequenced. The polymorphic characters and natural selection of the region were analysed using the DnaSP and MEGA4 programs. Results Thirty-two point mutations (28 non-synonymous and four synonymous mutations) were identified in PvDBPII among the Myanmar P. vivax isolates. Sequence analyses revealed that 12 different PvDBPII haplotypes were identified in Myanmar P. vivax isolates and that the region has evolved under positive natural selection. High selective pressure preferentially acted on regions identified as B- and T-cell epitopes of PvDBPII. Recombination may also be played a role in the resulting genetic diversity of PvDBPII. Conclusions PvDBPII of Myanmar P. vivax isolates displays a high level of genetic polymorphism and is under selective pressure. Myanmar P. vivax isolates share distinct types of PvDBPII alleles that are different from those of other geographical areas. These results will be useful for understanding the nature of the P. vivax population in Myanmar and for development of PvDBPII-based vaccine. PMID:22380592

Quantification of Fel d 1 in house dust samples of cat allergic patients by using monoclonal antibody specific to a novel IgE-binding epitope.

PubMed

Tasaniyananda, Natt; Tungtrongchitr, Anchalee; Seesuay, Watee; Sakolvaree, Yuwaporn; Aiumurai, Pisinee; Indrawattana, Nitaya; Chaicumpa, Wanpen; Sookrung, Nitat

2018-03-01

Avoidance of allergen exposure is an effective measure for preventing naÏve and allergic individuals from sensitization (primary intervention) and disease aggravation (secondary intervention), respectively. Regular monitoring of the allergens in the environment is required for the effective intervention. Thus, there is a need for cost-effective test kits for environmental allergen quantifications. To invent a test kit for quantification of cat major allergen, Fel d 1. A mouse monoclonal antibody (MAb) specific to the newly identified IgE-binding conformational epitope of the cat major allergen (Fel d 1) and rabbit polyclonal IgG to recombinant Fel d 1 were used as allergen capture and detection reagents, respectively. Native Fel d 1 was used in constructing a standard curve. Sixteen of 36 dust samples collected from houses of cat allergic subjects in Bangkok contained Fel d 1 above 0.29 μg/gram of dust which is considered as a novel threshold level for causing cat allergy sensitization or symptoms. Among them, 7 samples contained the allergen exceeding 2.35 μg/gram of dust which is the level that would aggravate asthma. Results of the allergen quantification using the locally made test kit showed strong correlation (r = 0.923) with the allergen quantification using commercialized reagents. The assay using MAb to Fel d 1 IgE-binding epitope of this study has potential application as an economic and practical tool for cat allergy intervention measure especially in localities where health resources are relatively limited.

Effect of heat treatment and enzymatic digestion on the B cell epitopes of cow's milk proteins.

PubMed

Morisawa, Y; Kitamura, A; Ujihara, T; Zushi, N; Kuzume, K; Shimanouchi, Y; Tamura, S; Wakiguchi, H; Saito, H; Matsumoto, K

2009-06-01

Processing milk leads to changes in clinical allergenicity. However, the mechanism by which heat treatment affects the allergenicity of milk proteins is not fully understood. We investigated the effect of heat treatment and enzymatic digestion on the allergenicity of B cell epitopes of milk proteins using a histamine release assay. Human basophils were passively sensitized using sera from 10 patients with allergies to cow's milk. All the patients experienced symptoms immediately after ingesting milk. The human basophils were obtained from umbilical cord blood mononuclear cells after culturing the mononuclear cells for 3-4 weeks in the presence of IL-3. After sensitization with 10% patient sera for 48 h, the cells were stimulated with untreated, heat-treated, or heat-treated and pepsin-and-trypsin-digested beta-lactoglobulin or alpha-casein for 1 h. The histamine concentrations in the supernatants were then measured by radioimmunoassay. Heat treatment alone did not alter the molecular weight of beta-lactoglobulin or alpha-casein. Heat treatment of beta-lactoglobulin significantly increased its susceptibility to enzymatic digestion in a time- and temperature-dependent manner and reduced its ability to induce histamine release from sensitized basophils. Similar findings were not observed for alpha-casein. The combination of heat treatment and enzymatic digestion reduced the abilities of both beta-lactoglobulin and alpha-casein to induce histamine release from passively sensitized basophils. Heat treatment reduced the allergenicity of beta-lactoglobulin by inducing conformational changes and by increasing its susceptibility to enzymatic digestion, both of which disrupted B cell epitopes, whereas heat treatment alone did not alter the allergenicity of alpha-casein.

Conformational changes in intact dengue virus reveal serotype-specific expansion

PubMed Central

Lim, Xin-Xiang; Chandramohan, Arun; Lim, Xin Ying Elisa; Bag, Nirmalya; Sharma, Kamal Kant; Wirawan, Melissa; Wohland, Thorsten; Lok, Shee-Mei; Anand, Ganesh S.

2017-01-01

Dengue virus serotype 2 (DENV2) alone undergoes structural expansion at 37 °C (associated with host entry), despite high sequence and structural homology among the four known serotypes. The basis for this differential expansion across strains and serotypes is unknown and necessitates mapping of the dynamics of dengue whole viral particles to describe their coordinated motions and conformational changes when exposed to host-like environments. Here we capture the dynamics of intact viral particles of two serotypes, DENV1 and DENV2, by amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and time resolved Förster Resonance Energy Transfer. Our results show temperature-dependent dynamics hotspots on DENV2 and DENV1 particles with DENV1 showing expansion at 40 °C but not at 37 °C. HDXMS measurement of virion dynamics in solution offers a powerful approach to identify potential epitopes, map virus-antibody complex structure and dynamics, and test effects of multiple host-specific perturbations on viruses and virus-antibody complexes. PMID:28186093

Genetic association of cyclooxygenase-2 gene polymorphisms with Parkinson's disease susceptibility in Chinese Han population.

PubMed

Dai, Yi; Wu, Yuquan; Li, Yansheng

2015-01-01

The aim of this study was to explore the genetic association of cyclooxygenase-2 (COX2) gene promoter region polymorphisms with Parkinson's disease (PD) susceptibility in Chinese Han population. The genotyping of COX2 gene polymorphisms was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 122 patients with PD and 120 healthy persons. The association strength of gene polymorphism with disease was measured by odds ratio (OR) and 95% confidence interval (95% CI) calculated using χ(2) test which also evaluated the Hardy-Weinberg equilibrium (HWE) of gene polymorphism in controls. The linkage disequilibrium and haplotype were also analyzed as evidence in the analysis of association. On condition that the genotypes distributions of COX2 -1290A>G, -1195G>A, -765G>C in the control group all conformed to HWE, however, only the homozygous genotype AA of -1195G>A polymorphism showed an association with PD (OR=0.432, 95% CI=0.196-0.950). In addition, in haplotype analysis, G-A-C haplotype frequency in cases was significantly lower than the controls, compared with the common haplotype A-G-G (P=0.031, OR=0.375, 95% CI=0.149-0.940). COX2 -1195G>A polymorphism might play a protective role in the onset of PD and G-A-C haplotype in this three promoter region polymorphisms also showed a negative association.

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

PubMed Central

Milton, Deanna L.; Schneck, Amy N.; Ziech, Dominique A.; Ba, Mariam; Facemyer, Kevin C.; Halayko, Andrew J.; Baker, Jonathan E.; Gerthoffer, William T.; Cremo, Christine R.

2011-01-01

The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber–associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells. PMID:21205888

Self-assembling Polypeptide Nanoparticles: Design, Synthesis, Biophysical Characterization and Biomedical Applications

NASA Astrophysics Data System (ADS)

Araujo Pereira Falcao Pimentel, Tais de

Inspired by the architecture of icosahedral viruses, self-assembling polypeptide nanoparticles (SAPN) with icosahedral symmetry were developed. The building block for the SAPN was a single polypeptide chain. Similarly, the capsid of quite a few small viruses are built from one single peptide chain. The polypeptide chain of the SAPN consists of a pentameric coiled-coil domain at the N-terminus joined by a short linker segment to a trimeric coiled-coil domain at the C-terminus. Here we have studied factors governing self-assembly of the SAPN such as linker constitution and trimer length. The interdomain linker 2i88 afforded the most homogenous nanoparticles as verified by TEM and DLS. Furthermore, AUC and STEM analyses suggest that the nanoparticles formed using the linker 2i88 have a T=3-like architecture confirming computer modeling predictions. As for trimer length, we have shown that it is possible to synthesize SAPN with a trimer that is as short as only 17 amino acids. Given that the N-terminus and C-terminus of the SAPN can be extended to include epitopes and give rise to a repetitive antigen display system, vaccine applications of the SAPN were also investigated here. We grafted parts of the SARS virus' spike protein onto our SAPN to repetitively display this B-cell epitope. Biophysical characterization showed that single nanoparticles of the expected size range were formed. Immunization experiments in mice at University of Colorado Denver revealed that the antibodies elicited were conformation-specific. Moreover, the antibodies significantly inhibited SARS virus infection of Vero E6 cells. SAPN were also functionalized at the C-terminus with a B-cell epitope from the circumsporozoite protein (CSP) of the malaria parasite Plasmodium falciparum and at the N-terminus with CTL epitopes from CSP. The trimeric coiled-coil domains of these malaria SAPN were modified to include a HTL epitope. Even will all these modifications, self-assembly occurred as confirmed by TEM and DLS. In immunization experiments performed at WRAIR good immune responses were obtained. Another biomedical application of SAPN is the development of a peptide-based serodiagnostic assay for tuberculosis (Tb). In an ELISA format, Tb-SAPN showed modest responses in serodiagnosis of Tb.

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

Genetic polymorphisms and protein structures in growth hormone, growth hormone receptor, ghrelin, insulin-like growth factor 1 and leptin in Mehraban sheep.

PubMed

Bahrami, A; Behzadi, Sh; Miraei-Ashtiani, S R; Roh, S-G; Katoh, K

2013-09-15

The somatotropic axis, the control system for growth hormone (GH) secretion and its endogenous factors involved in the regulation of metabolism and energy partitioning, has promising potentials for producing economically valuable traits in farm animals. Here we investigated single nucleotide polymorphisms (SNPs) of the genes of factors involved in the somatotropic axis for growth hormone (GH1), growth hormone receptor (GHR), ghrelin (GHRL), insulin-like growth factor 1 (IGF-I) and leptin (LEP), using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in 452 individual Mehraban sheep. A nonradioactive method to allow SSCP detection was used for genomic DNA and PCR amplification of six fragments: exons 4 and 5 of GH1; exon 10 of GH receptor (GHR); exon 1 of ghrelin (GHRL); exon 1 of insulin-like growth factor-I (IGF-I), and exon 3 of leptin (LEP). Polymorphisms were detected in five of the six PCR products. Two electrophoretic patterns were detected for GH1 exon 4. Five conformational patterns were detected for GH1 exon 5 and LEP exon 3, and three for IGF-I exon 1. Only GHR and GHRL were monomorphic. Changes in protein structures due to variable SNPs were also analyzed. The results suggest that Mehraban sheep, a major breed that is important for the animal industry in Middle East countries, has high genetic variability, opening interesting prospects for future selection programs and preservation strategies. Copyright © 2013 Elsevier B.V. All rights reserved.

Conformational dimorphism of isochroman-1-ones in the solid state

NASA Astrophysics Data System (ADS)

Babjaková, Eva; Hanulíková, Barbora; Dastychová, Lenka; Kuřitka, Ivo; Nečas, Marek; Vícha, Robert

2014-12-01

Isochroman-1-one derivatives, which are relatives of coumarins, display a broad spectrum of biological activity; therefore, these derivatives attract the attention of chemists. A series of new isochroman-1-ones were prepared by the reaction of benzyl-derived Grignard reagents with acyl chlorides. All of the prepared compounds were characterized using single-crystal X-ray diffraction as well as FT-IR, NMR and MS techniques. Single crystal X-ray diffraction analysis revealed that the isochromanones can adopt two distinct conformations in the solid state. For one of the compounds, two polymorphs with unique forms crystallized separately under different temperatures. The packing of all of the examined crystals is stabilized via weak intramolecular C-H⋯π and/or C-H⋯O interactions. Although the closed conformer was predominantly found in the actual crystals, the open conformer is thermochemically more stable for all of the examined compounds according to DFT calculations.

Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance.

PubMed

Theillet, François-Xavier; Frank, Martin; Vulliez-Le Normand, Brigitte; Simenel, Catherine; Hoos, Sylviane; Chaffotte, Alain; Bélot, Frédéric; Guerreiro, Catherine; Nato, Farida; Phalipon, Armelle; Mulard, Laurence A; Delepierre, Muriel

2011-12-01

Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments. Additionally, these complementary pieces of information give evidence to the ability of the studied mAb to recognize internal as well as terminal epitopes of its cognate polysaccharide antigen. Hence, we show that an appropriate combination of computational and experimental methods provides a basis to explore carbohydrate functional mimicry and receptor binding. The strategy may facilitate the design of either ligands or carbohydrate recognition domains, according to needed improvements of the natural carbohydrate:receptor properties. © The Author 2011. Published by Oxford University Press. All rights reserved.

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

PubMed

Boyles, Jeffrey S; Atwell, Shane; Druzina, Zhanna; Heuer, Josef G; Witcher, Derrick R

2016-11-01

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala 41 , Glu 44 , and His 45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859. © 2016 The Protein Society.

Human Immune Response to Outer Membrane Protein CD of Moraxella catarrhalis in Adults with Chronic Obstructive Pulmonary Disease

PubMed Central

Murphy, Timothy F.; Kirkham, Charmaine; Liu, Dai-Fang; Sethi, Sanjay

2003-01-01

Moraxella catarrhalis is a common cause of lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). The antibody response to outer membrane protein (OMP) CD, a highly conserved surface protein of M. catarrhalis under consideration as a vaccine antigen, was studied in adults with COPD following 40 episodes of infection or colonization. Following infection or colonization, 9 of 40 patients developed new serum immunoglobulin G (IgG) to OMP CD, as measured by enzyme-linked immunosorbent assay. Adsorption assays revealed that a proportion of the serum IgG was directed toward surface-exposed epitopes on OMP CD in six of the nine patients who developed new IgG to OMP CD. Immunoblot assays with fusion peptide constructs indicated that the new antibodies that developed after infection or colonization recognized conformational epitopes, particularly in the carboxy region of the protein. Three of 28 patients developed new mucosal IgA to OMP CD in sputum supernatants. This study establishes that OMP CD is a target of a systemic and mucosal immune response following infection and colonization in some patients with COPD. PMID:12595444

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

PubMed

Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

2014-12-02

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

Peptide selection and antibody generation for the prospective immunorecognition of Cry1Ab16 protein of transgenic maize.

PubMed

Costa, Joana; Marani, Mariela M; Grazina, Liliana; Villa, Caterina; Meira, Liliana; Oliveira, M Beatriz P P; Leite, José R S A; Mafra, Isabel

2017-09-15

The introduction of genes isolated from different Bacillus thuringiensis strains to express Cry-type toxins in transgenic crops is a common strategy to confer insect resistance traits. This work intended to extensively in silico analyse Cry1A(b)16 protein for the identification of peptide markers for the biorecognition of transgenic crops. By combining two different strategies based on several bioinformatic tools for linear epitope prediction, a set of seven peptides was successfully selected as potential Cry1A(b)16 immunogens. For the prediction of conformational epitopes, Cry1A(b)16 models were built on the basis of three independent templates of homologue proteins of Cry1A(a) and Cry1A(c) using an integrated approach. PcH_736-746 and PcH_876-886 peptides were selected as the best candidates, being synthesised and used for the production of polyclonal antibodies. To the best of our knowledge, this is the first attempt of selecting and defining linear peptides as immunogenic markers of Cry1A(b)-type toxins in transgenic maize. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nature vs. nurture in human sociality: multi-level genomic analyses of social conformity.

PubMed

Chen, Biqing; Zhu, Zijian; Wang, Yingying; Ding, Xiaohu; Guo, Xiaobo; He, Mingguang; Fang, Wan; Zhou, Qin; Zhou, Shanbi; Lei, Han; Huang, Ailong; Chen, Tingmei; Ni, Dongsheng; Gu, Yuping; Liu, Jianing; Rao, Yi

2018-05-01

Social conformity is fundamental to human societies and has been studied for more than six decades, but our understanding of its mechanisms remains limited. Individual differences in conformity have been attributed to social and cultural environmental influences, but not to genes. Here we demonstrate a genetic contribution to conformity after analyzing 1,140 twins and single-nucleotide polymorphism (SNP)-based studies of 2,130 young adults. A two-step genome-wide association study (GWAS) revealed replicable associations in 9 genomic loci, and a meta-analysis of three GWAS with a sample size of ~2,600 further confirmed one locus, corresponding to the NAV3 (Neuron Navigator 3) gene which encodes a protein important for axon outgrowth and guidance. Further multi-level (haplotype, gene, pathway) GWAS strongly associated genes including NAV3, PTPRD (protein tyrosine phosphatase receptor type D), ARL10 (ADP ribosylation factor-like GTPase 10), and CTNND2 (catenin delta 2), with conformity. Magnetic resonance imaging of 64 subjects shows correlation of activation or structural features of brain regions with the SNPs of these genes, supporting their functional significance. Our results suggest potential moderate genetic influence on conformity, implicate several specific genetic elements in conformity and will facilitate further research on cellular and molecular mechanisms underlying human conformity.

Molecular evolution of the leptin exon 3 in some species of the family Canidae

PubMed Central

Chmurzynska, Agata; Zajac, Magdalena; Switonski, Marek

2003-01-01

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical. PMID:12939206

Primary structure of Lep d I, the main Lepidoglyphus destructor allergen.

PubMed

Varela, J; Ventas, P; Carreira, J; Barbas, J A; Gimenez-Gallego, G; Polo, F

1994-10-01

The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N-terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non-conservative substitutions in three of them. No potential N-glycosylation site was revealed by peptide sequencing. The 125-residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.

The chemistry of prions: small molecules, protein conformers and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Background/Introduction. Prions propagate by converting a normal cellular isoform (PrPC) into the prion isoform (PrPSc) in a template-driven process. The lysines in PrPC are highly conserved and strongly influence prion propagation, based on studies using natural polymorphisms of PrPC and transg...

Association between mismatch repair gene MSH3 codons 1036 and 222 polymorphisms and sporadic prostate cancer in the Iranian population.

PubMed

Jafary, Fariba; Salehi, Mansoor; Sedghi, Maryam; Nouri, Nayereh; Jafary, Farzaneh; Sadeghi, Farzaneh; Motamedi, Shima; Talebi, Maede

2012-01-01

The mismatch repair system (MMR) is a post-replicative DNA repair mechanism whose defects can lead to cancer. The MSH3 protein is an essential component of the system. We postulated that MSH3 gene polymorphisms might therefore be associated with prostate cancer (PC). We studied MSH3 codon 222 and MSH3 codon 1036 polymorphisms in a group of Iranian sporadic PC patients. A total of 60 controls and 18 patients were assessed using the polymerase chain reaction and single strand conformational polymorphism. For comparing the genotype frequencies of patients and controls the chi-square test was applied. The obtained result indicated that there was significantly association between G/A genotype of MSH3 codon 222 and G/G genotype of MSH3 codon 1036 with an increased PC risk (P=0.012 and P=0.02 respectively). Our results demonstrated that MSH3 codon 222 and MSH3 codon 1036 polymorphisms may be risk factors for sporadic prostate cancer in the Iranian population.

A triclinic polymorph of tri­cyclo­hexyl­phosphane sulfide: crystal structure and Hirshfeld surface analysis

PubMed Central

Tan, Yi Jiun; Yeo, Chien Ing; Halcovitch, Nathan R.; Jotani, Mukesh M.

2017-01-01

The title compound, (C6H11)3PS (systematic name: tri­cyclo­hexyl-λ5-phosphane­thione), is a triclinic (P-1, Z′ = 1) polymorph of the previously reported ortho­rhom­bic form (Pnma, Z′ = 1/2) [Kerr et al. (1977 ▸). Can. J. Chem. 55, 3081–3085; Reibenspies et al. (1996 ▸). Z. Kristallogr. 211, 400]. While conformational differences exist between the non-symmetric mol­ecule in the triclinic polymorph, cf. the mirror-symmetric mol­ecule in the ortho­rhom­bic form, these differences are not chemically significant. The major feature of the mol­ecular packing in the triclinic polymorph is the formation of linear chains along the a axis sustained by methine-C—H⋯S(thione) inter­actions. The chains pack with no directional inter­actions between them. The analysis of the Hirshfeld surface for both polymorphs indicates a high degree of similarity, being dominated by H⋯H (ca 90%) and S⋯H/H⋯S contacts. PMID:28435705

Polymorphism complexity and handedness inversion in serum albumin amyloid fibrils.

PubMed

Usov, Ivan; Adamcik, Jozef; Mezzenga, Raffaele

2013-12-23

Protein-based amyloid fibrils can show a great variety of polymorphic structures within the same protein precursor, although the origins of these structural homologues remain poorly understood. In this work we investigate the fibrillation of bovine serum albumin--a model globular protein--and we follow the polymorphic evolution by a statistical analysis of high-resolution atomic force microscopy images, complemented, at larger length scales, by concepts based on polymer physics formalism. We identify six distinct classes of coexisting amyloid fibrils, including flexible left-handed twisted ribbons, rigid right-handed helical ribbons and nanotubes. We show that the rigid fibrils originate from flexible fibrils through two diverse polymorphic transitions, first, via a single-fibril transformation when the flexible left-handed twisted ribbons turn into the helical left-handed ribbons, to finally evolve into nanotube-like structures, and second, via a double-fibril transformation when two flexible left-handed twisted ribbons wind together resulting in a right-handed twisted ribbon, followed by a rigid right-handed helical ribbon polymorphic conformation. Hence, the change in handedness occurs with an increase in the level of the fibril's structural organization.

Ala397Asp mutation of myosin VIIA gene segregating in a Spanish family with type-Ib Usher syndrome.

PubMed

Espinós, C; Millán, J M; Sánchez, F; Beneyto, M; Nájera, C

1998-06-01

In the current study, 12 Spanish families affected by type-I Usher syndrome, that was previously linked to chromosome 11q, were screened for the presence of mutations in the N-terminal coding portion of the motor domain of the myosin VIIA gene by single-strand conformation polymorphism analysis of the first 14 exons. A mutation (Ala397Asp) segregating with the disease was identified, and several polymorphisms were also detected. It is presumed that the other USHIB mutations in these families could be located in the unscreened regions of the gene.

Molecular and immunohistochemical analysis of P53 in phaeochromocytoma.

PubMed Central

Dahia, P. L.; Aguiar, R. C.; Tsanaclis, A. M.; Bendit, I.; Bydlowski, S. P.; Abelin, N. M.; Toledo, S. P.

1995-01-01

We searched for mutations of the p53 gene in 25 phaeochromocytomas using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the entire conserved region of the gene, encompassing exons 4-8; expression of the p53 protein was assessed by immunohistochemistry. No mutations were found, while a polymorphism in codon 72 was observed. Immunohistochemistry revealed nuclear p53 overexpression in one tumour sample. We conclude that mutations of the 'hotspot' region of the p53 gene do not seem to play a role in the pathogenesis of phaeochromocytoma. Images Figure 1 Figure 2 Figure 3 PMID:7577469

(E)-Ethyl 3-(3,4-dihydroxyphenyl)prop-2-enoate: a natural polymorph extracted from Aristotelia chilensis (Maqui).

PubMed

Paz, Cristian; Moreno, Yanko; Becerra, José; Silva, Mario; Burgos, Viviana; Freire, Eleonora; Baggio, Ricardo

2013-07-01

The natural title compound, C11H12O4, extracted from the Chilean native tree Aristotelia chilensis (Maqui), is a polymorph of the synthetic E form reported by Xia, Hu & Rao [Acta Cryst. (2004), E60, o913-o914]. Both rotational conformers are identical from a metrical point of view, and only differ in the orientation of the 3,4-dihydroxyphenyl ring with respect to the rest of the molecule, which leads to completely different crystal structure arrangements and packing efficiencies. The reasons behind both reside in the different hydrogen-bonding interactions.

Isolation and characterization of 21 polymorphic microsatellite loci in the Japanese dace (Tribolodon hakonensis)

USGS Publications Warehouse

Koizumi, Noriyuki; Quinn, Thomas W.; Park, Myeongsoo; Fike, Jennifer A.; Nishida, Kazuya; Takemura, Takeshi; Watabe, Keiji; Mori, Atsushi

2011-01-01

Twenty one polymorphic microsatellite loci for the Japanese dace (Tribolodon hakonensis) were isolated and characterized. The number of observed alleles per locus in 32 individuals ranged from 3 to 30. The observed and expected heterozygosities ranged from 0.125 to 0.969 and from 0.175 to 0.973, respectively. All loci conformed to Hardy–Weinberg equilibrium, no linkage disequilibrium was observed between pairs of loci and no loci showed evidence of null alleles. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.

Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

PubMed Central

Sircar, Gaurab; Saha, Bodhisattwa; Mandal, Rahul Shubhra; Pandey, Naren; Saha, Sudipto; Gupta Bhattacharya, Swati

2015-01-01

Background Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’. Method The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. Results The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens. Conclusion/Significance The present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy. PMID:26672984

Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates.

PubMed

Kang, Jung-Mi; Lee, Jinyoung; Moe, Mya; Jun, Hojong; Lê, Hương Giang; Kim, Tae Im; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Shin, Ho-Joon; Kim, Tong-Soo; Na, Byoung-Kuk

2018-02-07

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the strong selective pressure of host immunity on the PfAMA-1 gene. These results have significant implications in understanding the nature of Myanmar PfAMA-1 along with global PfAMA-1. They also provide useful information for the development of effective malaria vaccine based on this antigen.

Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol

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

Kadam, Rameshwar U.; Wilson, Ian A.

The broad-spectrum antiviral drug Arbidol shows efficacy against influenza viruses by targeting the hemagglutinin (HA) fusion machinery. However, the structural basis of the mechanism underlying fusion inhibition by Arbidol has remained obscure, thereby hindering its further development as a specific and optimized influenza therapeutic. We determined crystal structures of Arbidol in complex with influenza virus HA from pandemic 1968 H3N2 and recent 2013 H7N9 viruses. Arbidol binds in a hydrophobic cavity in the HA trimer stem at the interface between two protomers. This cavity is distal to the conserved epitope targeted by broadly neutralizing stem antibodies and is ~16 Åmore » from the fusion peptide. Arbidol primarily makes hydrophobic interactions with the binding site but also induces some conformational rearrangements to form a network of inter- and intraprotomer salt bridges. By functioning as molecular glue, Arbidol stabilizes the prefusion conformation of HA that inhibits the large conformational rearrangements associated with membrane fusion in the low pH of the endosome. This unique binding mode compared with the small-molecule inhibitors of other class I fusion proteins enhances our understanding of how small molecules can function as fusion inhibitors and guides the development of broad-spectrum therapeutics against influenza virus.« less

Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively

PubMed Central

Orriss, George L.; Niland, Stephan; Johanningmeier, Benjamin; Pohlentz, Gottfried; Meier, Markus; Karrasch, Simone; Estevão-Costa, Maria Inacia; Martins Lima, Augusto; Stetefeld, Jörg

2017-01-01

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin. PMID:28704364

Tissue Specificity of Human Angiotensin I-Converting Enzyme

PubMed Central

Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

2015-01-01

Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

ACE phenotyping in Gaucher disease.

PubMed

Danilov, Sergei M; Tikhomirova, Victoria E; Metzger, Roman; Naperova, Irina A; Bukina, Tatiana M; Goker-Alpan, Ozlem; Tayebi, Nahid; Gayfullin, Nurshat M; Schwartz, David E; Samokhodskaya, Larisa M; Kost, Olga A; Sidransky, Ellen

2018-04-01

Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen. The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease. Copyright © 2018 Elsevier Inc. All rights reserved.

A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

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

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

A Dominant Conformational Role for Amino Acid Diversity in Minimalist Protein-Protein Interfaces

PubMed Central

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

2008-01-01

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies”. One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose binding protein (MBP). The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution x-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side-chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces. PMID:18602117

Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

PubMed

Souza, M T; Carvalho-Zilse, G A

2014-07-25

In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species.

Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox.

PubMed

Kaever, Thomas; Matho, Michael H; Meng, Xiangzhi; Crickard, Lindsay; Schlossman, Andrew; Xiang, Yan; Crotty, Shane; Peters, Bjoern; Zajonc, Dirk M

2016-05-01

Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen. Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an attempt to better understand the interplay between the antibodies and their antigens, we generated and functionally characterized a panel of anti-A27 antibodies and studied their interaction with the epitope using X-ray crystallography. We identified one protective antibody that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding. Analysis of the antibody-antigen interaction supports a model in which antibodies that can interfere with the functional activity of the antigen are more likely to confer protection than those that bind at the extremities of the antigen. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dengue Virus Type 3 Adaptive Changes during Epidemics in São Jose de Rio Preto, Brazil, 2006–2007

PubMed Central

Bosch, Irene; Schimitt, Diane; Calzavara-Silva, Carlos E.; de A Zanotto, Paolo M.; Nogueira, Maurício L.

2013-01-01

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak. PMID:23667626

Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

PubMed Central

Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

2016-01-01

X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

FT-IR spectra of the anti-HIV nucleoside analogue d4T (Stavudine). Solid state simulation by DFT methods and scaling by different procedures

NASA Astrophysics Data System (ADS)

Alcolea Palafox, M.; Kattan, D.; Afseth, N. K.

2018-04-01

A theoretical and experimental vibrational study of the anti-HIV d4T (stavudine or Zerit) nucleoside analogue was carried out. The predicted spectra in the three most stable conformers in the biological active anti-form of the isolated state were compared. Comparison of the conformers with those of the natural nucleoside thymidine was carried out. The calculated spectra were scaled by using different scaling procedures and three DFT methods. The TLSE procedure leads to the lowest error and is thus recommended for scaling. With the population of these conformers the IR gas-phase spectra were predicted. The crystal unit cell of the different polymorphism forms of d4T were simulated through dimer forms by using DFT methods. The scaled spectra of these dimer forms were compared. The FT-IR spectrum was recorded in the solid state in the 400-4000 cm-1 range. The respective vibrational bands were analyzed and assigned to different normal modes of vibration by comparison with the scaled vibrational values of the different dimer forms. Through this comparison, the polymorphous form of the solid state sample was identified. The study indicates that d4T exist only in the ketonic form in the solid state. The results obtained were in agreement with those determined in related anti-HIV nucleoside analogues.

Conformational dimorphism in o-nitrobenzoic acid: alternative ways to avoid the O...O clash.

PubMed

Ibragimov, Aziz; Ashurov, Jamshid; Ibragimov, Bakhtiyar; Wang, Ai; Mouhib, Halima; Englert, Ulli

2016-07-01

Polymorphism is a challenging phenomenon and the competitive packing alternatives which are characteristic for polymorphs may be encountered for essentially rigid molecules. A second crystal form of the well known compound o-nitrobenzoic acid, C7H5NO4, an important intermediate in the production of dyes, pharmaceuticals and agrochemicals, is described. Although obtained serendipitously, its intra- and intermolecular features match expectations from database searches and theoretical calculations. O-H...O hydrogen-bonded carboxylic acid dimers represent the building blocks in both polymorphs. For steric reasons and in agreement with a calculated potential energy surface, the carboxylic acid and nitro groups cannot simultaneously be coplanar with the benzene ring but have to tilt. In the well established crystal form, this out-of-plane torsion is more pronounced for the nitro substituent. In contrast, the new polymorph is characterized by a major tilt of the carboxylic acid group. The molecules in both alternative crystal forms achieve a similar compromise with respect to acceptable intramolecular O...O contacts.