Human leukemia antigen-A*0201-restricted epitopes of human endogenous retrovirus W family envelope (HERV-W env) induce strong cytotoxic T lymphocyte responses.

PubMed

Tu, Xiaoning; Li, Shan; Zhao, Lijuan; Xiao, Ran; Wang, Xiuling; Zhu, Fan

2017-08-01

Human endogenous retrovirus W family (HERV-W) envelope (env) has been reported to be related to several human diseases, including autoimmune disorders, and it could activate innate immunity. However, there are no reports investigating whether human leukemia antigen (HLA)-A*0201 + restriction is involved in the immune response caused by HERV-W env in neuropsychiatric diseases. In the present study, HERV-W env-derived epitopes presented by HLA-A*0201 are described with the potential for use in adoptive immunotherapy. Five peptides displaying HLA-A*0201-binding motifs were predicted using SYFEPITHI and BIMAS, and synthesized. A CCK-8 assay showed peptides W, Q and T promoted lymphocyte proliferation. Stimulation of peripheral blood mononuclear cells from HLA-A*0201 + donors with each of these peptides induced peptide-specific CD8 + T cells. High numbers of IFN-γ-secreting T cells were also detectable after several weekly stimulations with W, Q and T. Besides lysis of HERV-W env-loaded target cells, specific apoptosis was also observed. These data demonstrate that human T cells can be sensitized toward HERV-W env peptides (W, Q and T) and, moreover, pose a high killing potential toward HERV-W env-expressing U251 cells. In conclusion, peptides W Q and T, which are HERV-W env antigenic epitopes, have both antigenicity and immunogenicity, and can cause strong T cell immune responses. Our data strengthen the view that HERV-W env should be considered as an autoantigen that can induce autoimmunity in neuropsychiatric diseases, such as multiple sclerosis and schizophrenia. These data might provide an experimental foundation for a HERV-W env peptide vaccine and new insight into the treatment of neuropsychiatric diseases.

Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor

NASA Astrophysics Data System (ADS)

Kumar, Amit; Delogu, Francesco

2017-02-01

The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR’s cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient.

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

NASA Astrophysics Data System (ADS)

Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

2013-03-01

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses.

PubMed

Blyth, Emily; Clancy, Leighton; Simms, Renee; Gaundar, Shivashni; O'Connell, Philip; Micklethwaite, Kenneth; Gottlieb, David J

2011-11-27

BK virus (BKV) infection causes hemorrhagic cystitis posthemopoietic stem-cell transplant and graft loss in renal transplant recipients. Reactivation occurs in up to 60% of patients in both groups. Treatment-related cellular immunosuppression is a major contributor to the development of BKV-related disease, but the targets of the immune response are not well characterized. Immunotherapy by adoptive transfer of cellular effectors has been shown to be effective in controlling and preventing some virus-related diseases in transplant recipients, particularly Epstein-Barr virus and cytomegalovirus. Infusion of BKV-specific T cells may potentially reconstitute functional BKV immunity and reduce clinical complications of BKV infection. BKV-specific T cells for clinical use in adoptive immunotherapy were generated using monocyte-derived dendritic cells pulsed with overlapping peptide mixes spanning the five BKV proteins VP1, VP2, VP3, large T antigen, and small T antigen. Phenotypic and functional characteristics of the cells were investigated as well as their antigen specificity. Expanded CD4(+) and CD8(+) cells responded to restimulation with BKV peptides principally from VP1, large T, or small T antigens; produced multiple cytokines; and showed cytotoxic activity against antigen-coated targets. Possible clinical uses for BKV-specific T cells generated using this method include immune reconstitution posthemopoietic stem-cell transplantation or prophylaxis and treatment of immune deficiency in renal transplant recipients, fulfilling the need for effective therapy for BKV-related hemorrhagic cystitis and renal dysfunction.

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax.

PubMed

Oscherwitz, Jon; Feldman, Daniel; Yu, Fen; Cease, Kemp B

2015-01-09

Anthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2-2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans. To optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants. Immunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits. An LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax. Copyright © 2014 Elsevier Ltd. All rights reserved.

Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

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

Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances themore » flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.« less

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

68Ga-Labeled Anti-Prostate-Specific Membrane Antigen Peptide as Marker for Androgen Deprivation Therapy Response in Prostate Cancer.

PubMed

Schlenkhoff, Carl Diedrich; Gaertner, Florian; Essler, Markus; Hauser, Stefan; Ahmadzadehfar, Hojjat

2016-05-01

Prostate cancer was diagnosed in a 71-year-old man with an elevated prostate-specific antigen. The CT of the abdomen showed multiple para-aortal lymph nodes, and thus, a Ga anti-prostate-specific membrane antigen (PSMA-11) PET/CT was initiated, which showed, aside from the prostate cancer and multiple iliacal and para-aortal lymph node metastases, an increased tracer uptake in a lymph node left cervical. According to this advanced disease, a palliative therapy with GnRH agonist was initiated. A second PSMA-11 PET/CT was performed 4 months later, which showed a very good response; thus, additional radiation of the pelvis and the draining lymphatic system was performed.

Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

NASA Astrophysics Data System (ADS)

Peri, Claudio; Gori, Alessandro; Gagni, Paola; Sola, Laura; Girelli, Daniela; Sottotetti, Samantha; Cariani, Lisa; Chiari, Marcella; Cretich, Marina; Colombo, Giorgio

2016-09-01

Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria.

Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.

PubMed

Jordan, Kimberly R; McMahan, Rachel H; Kemmler, Charles B; Kappler, John W; Slansky, Jill E

2010-03-09

Peptide vaccines enhance the response of T cells toward tumor antigens and represent a strategy to augment antigen-independent immunotherapies of cancer. However, peptide vaccines that include native tumor antigens rarely prevent tumor growth. We have assembled a set of peptide variants for a mouse-colon tumor model to determine how to improve T-cell responses. These peptides have similar affinity for MHC molecules, but differ in the affinity of the peptide-MHC/T-cell receptor interaction with a tumor-specific T-cell clone. We systematically demonstrated that effective antitumor responses are generated after vaccination with variant peptides that stimulate the largest proportion of endogenous T cells specific for the native tumor antigen. Importantly, we found some variant peptides that strongly stimulated a specific T-cell clone in vitro, but elicited fewer tumor-specific T cells in vivo, and were not protective. The T cells expanded by the effective vaccines responded to the wild-type antigen by making cytokines and killing target cells, whereas most of the T cells expanded by the ineffective vaccines only responded to the peptide variants. We conclude that peptide-variant vaccines are most effective when the peptides react with a large responsive part of the tumor-specific T-cell repertoire.

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

PubMed

Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

2004-02-01

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

Immune response induced by Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides in current and past infectious mononucleosis: a risk for multiple sclerosis?

PubMed

Mameli, G; Madeddu, G; Cossu, D; Galleri, G; Manetti, R; Babudieri, S; Mura, M Stella; Sechi, L A

2016-01-01

Infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) has been associated with increased risk of multiple sclerosis (MS). However, the mechanism linking these pathologies is unclear. Different reports indicate the association of EBV, and recently Mycobacterium avium subsp. paratuberculosis (MAP), with MS. For a better understanding of the role of these pathogens, the host response induced by selected antigenic peptides in subjects with a history of IM that significantly increases the risk of MS was investigated. Both humoral and cell-mediated response against peptides able to induce a specific immune activation in MS patients deriving from lytic and latent EBV antigens BOLF1(305-320), EBNA1(400-413), from MAP MAP_4027(18-32), MAP_0106c(121-132) and from human proteins IRF5(424-434) and MBP(85-98) in subjects with current and past IM were examined. EBNA1 and MAP_0106c peptides were able to induce a humoral immune response in subjects with a history of clinical IM in an independent manner. Moreover, these peptides were capable of inducing pro-inflammatory cytokine interferon γ by CD4+ and CD8+ T lymphocytes and interleukin 6 and tumour necrosis factor α by CD14+ monocyte cells. Our results highlight that EBV and MAP may be involved independently in the same causal process leading to MS in subjects with a history of IM. © 2015 EAN.

Humoral Responses to Diverse Autoimmune Disease-Associated Antigens in Multiple Sclerosis.

PubMed

Malyavantham, Kishore; Weinstock-Guttman, Bianca; Suresh, Lakshmanan; Zivadinov, Robert; Shanahan, Thomas; Badgett, Darlene; Ramanathan, Murali

2015-01-01

To compare frequencies of autoreactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression. The study analyzed 969 serum samples from 315 HC, 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114(Glc) and KIR4.1a and KIR4.1b and against 24 key endogenous antigens linked to diseases such as vasculitis, systemic sclerosis, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, polymyositis, scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease and primary biliary cirrhosis. Associations with disability and MRI measures of lesional injury and neurodegeneration were assessed. The frequencies of anti-KIR4.1a and anti-KIR4.1b peptide IgG positivity were 9.8% and 11.4% in HC compared to 4.9% and 7.5% in RR-MS, 8.6% for both peptides in SP-MS and 6.1% for both peptides in PP-MS (p = 0.13 for KIR4.1a and p = 0.34 for KIR4.1b), respectively. Antibodies against CSF114(Glc), KIR4.1a and KIR4.1b peptides were not associated with MS compared to HC, or with MS disease progression. HLA DRB1*15:01 positivity and anti-Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies. Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse autoreactive antibodies investigated are similar in MS and HC.

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

PubMed Central

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

2016-01-01

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

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...

Nonclassical T Cells and Their Antigens in Tuberculosis

PubMed Central

De Libero, Gennaro; Singhal, Amit; Lepore, Marco; Mori, Lucia

2014-01-01

T cells that recognize nonpeptidic antigens, and thereby are identified as nonclassical, represent important yet poorly characterized effectors of the immune response. They are present in large numbers in circulating blood and tissues and are as abundant as T cells recognizing peptide antigens. Nonclassical T cells exert multiple functions including immunoregulation, tumor control, and protection against infections. They recognize complexes of nonpeptidic antigens such as lipid and glycolipid molecules, vitamin B2 precursors, and phosphorylated metabolites of the mevalonate pathway. Each of these antigens is presented by antigen-presenting molecules other than major histocompatibility complex (MHC), including CD1, MHC class I–related molecule 1 (MR1), and butyrophilin 3A1 (BTN3A1) molecules. Here, we discuss how nonclassical T cells participate in the recognition of mycobacterial antigens and in the mycobacterial-specific immune response. PMID:25059739

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses

PubMed Central

Ma, Wenxue; Chen, Mingshui; Kaushal, Sharmeela; McElroy, Michele; Zhang, Yu; Ozkan, Cengiz; Bouvet, Michael; Kruse, Carol; Grotjahn, Douglas; Ichim, Thomas; Minev, Boris

2012-01-01

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund’s adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of −15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund’s adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo. PMID:22619507

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses.

PubMed

Ma, Wenxue; Chen, Mingshui; Kaushal, Sharmeela; McElroy, Michele; Zhang, Yu; Ozkan, Cengiz; Bouvet, Michael; Kruse, Carol; Grotjahn, Douglas; Ichim, Thomas; Minev, Boris

2012-01-01

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96(®) Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund's adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of -15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund's adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo.

Borrelia burgdorferi-specific IgA in Lyme Disease.

PubMed

D'Arco, Christina; Dattwyler, Raymond J; Arnaboldi, Paul M

2017-05-01

The laboratory diagnosis of Lyme disease is currently dependent on the detection of IgM and IgG antibodies against Borrelia burgdorferi, the causative agent of the disease. The significance of serum IgA against B. burgdorferi remains unclear. The production of intrathecal IgA has been noted in patients with the late Lyme disease manifestation, neuroborreliosis, but production of antigen-specific IgA during early disease has not been evaluated. In the current study, we assessed serum IgA binding to the B. burgdorferi peptide antigens, C6, the target of the FDA-cleared C6 EIA, and FlaB(211-223)-modVlsE(275-291), a peptide containing a Borrelia flagellin epitope linked to a modified VlsE sequence, in patients with early and late Lyme disease. Specific IgA was detected in 59 of 152 serum samples (38.8%) from early Lyme disease patients. Approximately 50% of early Lyme disease patients who were seropositive for peptide-specific IgM and/or IgG were also seropositive for peptide-specific IgA. In a subpopulation of patients, high peptide-specific IgA could be correlated with disseminated disease, defined as multiple erythema migrans lesions, and neurological disease complications. These results suggest that there may be an association between elevated levels of antigen-specific IgA and particular disease manifestations in some patients with early Lyme disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Peptide assemblies: from cell scaffolds to immune adjuvants

NASA Astrophysics Data System (ADS)

Collier, Joel

2011-03-01

This talk will discuss two interrelated aspects of peptide self-assemblies in biological applications: their use as matrices for regenerative medicine, and their use as chemically defined adjuvants for directing immune responses against engineered antigens. In the first half of the presentation, the design of peptide self-assemblies as analogues for the extracellular matrix will be described, with a focus on self-assemblies displaying multiple different cell-binding peptides. We conducted multi-factorial investigations of peptide co-assemblies containing several different ligand-bearing peptides using statistical ``design of experiments'' (DoE). Using the DoE techniques of factorial experimentation and response surface modeling, we systematically explored how precise combinations of ligand-bearing peptides modulated endothelial cell growth, in the process finding interactions between ligands not previously appreciated. By investigating immune responses against the materials intended for tissue engineering applications, we discovered that the basic self-assembling peptides were minimally immunogenic or non-immunogenic, even when delivered in strong adjuvants. -But when they were appended to an appropriately restricted epitope peptide, these materials raised strong and persistent antibody responses. These responses were dependent on covalent conjugation between the epitope and self-assembling domains of the peptides, were mediated by T cells, and could be directed towards both peptide epitopes and conjugated protein antigens. In addition to their demonstrated utility as scaffolds for regenerative medicine, peptide self-assemblies may also be useful as chemically defined adjuvants for vaccines and immunotherapies. This work was funded by NIH/NIDCR (1 R21 DE017703-03), NIH/NIBIB (1 R01 EB009701-01), and NSF (CHE-0802286).

Antigen-specific therapy of Graves´ disease and orbitopathy by induction of tolerance.

PubMed

Ungerer, Martin; Fabbender, Julia; Holthoff, Hans-Peter

2018-06-01

Graves´ disease is an autoimmune disorder, which is characterized by stimulatory antibodies targeting the human thyrotropin receptor (TSHR), resulting in hyperthyroidism and multiple organ damage. The disease can be modelled in mice using adenoviral immunizations with the extracellular A subunit of the TSHR, which induces a long-term stable disease state. TSHR binding cAMP-stimulatory antibodies, thyroid enlargement, elevated serum thyroxin levels, tachycardia, cardiac hypertrophy and orbitopathy are observed in these Ad-TSHR-immunized mice. T cell epitope-derived linear peptides have been identified using immunized HLA-DR3 transgenic mice, which may induce tolerance towards TSHR. A combination of such peptides are being investigated in a first clinical phase I trial in patients with Graves´ disease. Alternatively, intravenous administration of cyclic peptides derived from the interaction site of the TSHR A domain with stimulatory anti-TSHR antibodies can re-establish tolerance towards the antigen in immunized mice, improving symptoms of Graves´ disease within 3 - 4 months after starting these therapies. In immunologically naïve mice, administration of the cyclic peptides did not induce any immune response.

Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex*

PubMed Central

Yanaka, Saeko; Ueno, Takamasa; Shi, Yi; Qi, Jianxun; Gao, George F.; Tsumoto, Kouhei; Sugase, Kenji

2014-01-01

In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8+ T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition. PMID:25028510

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

PubMed

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

2016-02-01

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

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines.

PubMed

Jordan, Kimberly R; Buhrman, Jonathan D; Sprague, Jonathan; Moore, Brandon L; Gao, Dexiang; Kappler, John W; Slansky, Jill E

2012-10-01

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

PubMed

Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

2005-10-28

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

PubMed Central

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2. PMID:24223975

Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

PubMed

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets.

PubMed

Nielsen, Morten; Andreatta, Massimo

2016-03-30

Binding of peptides to MHC class I molecules (MHC-I) is essential for antigen presentation to cytotoxic T-cells. Here, we demonstrate how a simple alignment step allowing insertions and deletions in a pan-specific MHC-I binding machine-learning model enables combining information across both multiple MHC molecules and peptide lengths. This pan-allele/pan-length algorithm significantly outperforms state-of-the-art methods, and captures differences in the length profile of binders to different MHC molecules leading to increased accuracy for ligand identification. Using this model, we demonstrate that percentile ranks in contrast to affinity-based thresholds are optimal for ligand identification due to uniform sampling of the MHC space. We have developed a neural network-based machine-learning algorithm leveraging information across multiple receptor specificities and ligand length scales, and demonstrated how this approach significantly improves the accuracy for prediction of peptide binding and identification of MHC ligands. The method is available at www.cbs.dtu.dk/services/NetMHCpan-3.0 .

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

PubMed

Brune, Karl D; Buldun, Can M; Li, Yuanyuan; Taylor, Iona J; Brod, Florian; Biswas, Sumi; Howarth, Mark

2017-05-17

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PubMed

Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

2017-01-15

The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

Screening of Peptide Libraries Against Protective Antigen of Bacillus Anthracis in a Disposable Microfluidic Cartridge

DTIC Science & Technology

2011-11-28

New Reprint Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge W911NF-09-D-0001...against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Report Title ABSTRACT See attached. Screening of Peptide...Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Joshua M. Kogot1, Yanting Zhang2, Stephen J. Moore3

Antigenic properties of HCMV peptides displayed by filamentous bacteriophages vs. synthetic peptides.

PubMed

Ulivieri, Cristina; Citro, Alessandra; Ivaldi, Federico; Mascolo, Dina; Ghittoni, Raffaella; Fanigliulo, Daniela; Manca, Fabrizio; Baldari, Cosima Tatiana; Li Pira, Giuseppina; Del Pozzo, Giovanna

2008-08-15

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Vaccination with M2e-Based Multiple Antigenic Peptides: Characterization of the B Cell Response and Protection Efficacy in Inbred and Outbred Mice

PubMed Central

Wolf, Amaya I.; Mozdzanowska, Krystyna; Williams, Katie L.; Singer, David; Richter, Monique; Hoffmann, Ralf; Caton, Andrew J.; Otvos, Laszlo; Erikson, Jan

2011-01-01

Background The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains. Methodology/Principal Findings Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs. Conclusion/Significance Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population. PMID:22180783

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice.

PubMed

Wolf, Amaya I; Mozdzanowska, Krystyna; Williams, Katie L; Singer, David; Richter, Monique; Hoffmann, Ralf; Caton, Andrew J; Otvos, Laszlo; Erikson, Jan

2011-01-01

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains. Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs. Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

PubMed

Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

2012-11-01

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

PubMed Central

Haug, Markus; Brede, Gaute; Håkerud, Monika; Nedberg, Anne Grete; Gederaas, Odrun A.; Flo, Trude H.; Edwards, Victoria T.; Selbo, Pål K.; Høgset, Anders; Halaas, Øyvind

2018-01-01

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses. PMID:29670624

Development of Bacterial Display Peptides for use in Biosensing Applications

DTIC Science & Technology

2012-09-01

performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB...reagent, affinity reagent, bacterial display, multi-scale modeling, docking, protective antigen , SEB, biosensing 16. SECURITY CLASSIFICATION OF: 17...performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be

A biochip-based combined immunoassay for detection of serological status of Borrelia burgdorferi in Lyme borreliosis.

PubMed

Huang, Na-Li; Ye, Lei; Lv, Hui; Du, Yi-Xin; Schneider, Marion; Fan, Li-Bin; Du, Wei-Dong

2017-09-01

Dithiobis (succinimidyl undecanoate) modified gold surface biochip were used as a combined immunoassay platform for concurrently detecting immune responses to Borrelia burgdorferi (B. burgdorferi) sensu lato antigens, flagellin, outer surface protein C, variable major protein-like sequence proteins, and 3 VlsE protein IR 6 peptides. The peptides represented intrinsic Borrelia genospecies: B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. Fourier transform infrared spectroscopy was utilized to validate the surface chemical characteristics on the modified gold surface. The limits in detection of IgG antibody on the biochips were as little as 0.39μg/ml for anti-VlsE and 0.78μg/ml for anti-flagellin and anti-OspC, respectively. Samples from 56 neuroborreliosis (NB) patients and 114 healthy individuals were analyzed by the combined biochip. We found that the seroprevalences of IgM or IgG antibody against the 6 antigens were contributed to increased overall sensitivity by the multiplex immunobiochip assay. Serum combined positive rates of the 6 antigens in the patients were 92.86% for IgM antibody and 91.07% for IgG antibody. Part of the patients bore antibody responses against the 3 VlsE IR 6 variant peptides, indicating that Lyme borreliosis would attribute to consequence of multiple infections by one or more Borrelia burgdorferi strains. Concurrent assessment for both IgM and IgG antibodies against the protein antigens and B. burgdorferi IR 6 peptides in the sera of NB patients was beneficial from the biochip format, enabling detection of expanded serologic infection status and therapy strategy-making more efficiently. The combined biochip-based immunoassay, as a potential substitution of ELISA, provided a promising approach to extend the detection spectrum of infectious antibodies against a panel of Borrelia antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Prediction of Brugia malayi antigenic peptides: candidates for synthetic vaccine design against lymphatic filariasis.

PubMed

Gomase, Virendra S; Chitlange, Nikhilkumar R; Changbhale, Smruti S; Kale, Karbhari V

2013-08-01

Brugia malayi is a threadlike nematode cause's swelling of lymphatic organs, condition well known as lymphatic filariasis; till date no invention made to effectively address lymphatic filariasis. In this analysis we a have predicted suitable antigenic peptides from Brugia malayi antigen protein for peptide vaccine design against lymphatic filariasis based on cross protection phenomenon as, an ample immune response can be generated with a single protein subunit. We found MHC class II binding peptides of Brugia malayi antigen protein are important determinant against the diseased condition. The analysis shows Brugia malayi antigen protein having 505 amino acids, which shows 497 nonamers. In this assay, we have predicted MHC-I binding peptides for 8mer_H2_Db (optimal score- 15.966), 9mer_H2_Db (optimal score- 15.595), 10mer_H2_Db (optimal score- 19.405), 11mer_H2_Dballeles (optimal score- 23.801). We also predicted the SVM based MHCII-IAb nonamers, 51-FQQIDPLDA, 442-FAAIACLVH, 206-YLNPFGHQF, 167-WYVIMAACY, 367-YAMIVIRLL, 434- LVITTAANF, 176-LDSYCLWKP, 435-VITTAANFA, 364-WPGYAMIVI (optimal score- 13.963); MHCII-IAd nonamers, 52-QQIDPLDAE, 171-MAACYLDSY, 239-QWRSVILCN, 168-YVIMAACYL, 3-QYLSVHSLS, 322-EILLHAKVV, 417- LGIIASFVS, 396-KAIFLAHFG, 167-WYVIMAACY, 269-LALHCINVI, 93-FINKAAPKQ, 259-NCIIVLKAF, 79- QGVLLIIPR, 22-TILQRSQAI, 63-RGFVYGNVS, 109-NISSLAFET,(optimal score- 16.748); and MHCII-IAg7 nonamers 171-MAACYLDSY, 73-KIVNGAQGV, 259-NCIIVLKAF, 209-PFGHQFSFE, 102-SCDTLLKNI, 25-QRSQAIRIV, 444- AIACLVHLF, 88-SLVNGFINK, 252-FPRHQLLNC, 471-RFVLANDNE, 52-QQIDPLDAE, 469-HRRFVLAND, 457- SNRHYFLAD, 362-KSWPGYAMI, 476-NDNEGEDFE, 370-IVIRLLQAL (optimal score- 19.847) which represents potential binders from Brugia malayi antigen protein. The method integrates prediction of MHC class I binding proteasomal C-terminal cleavage peptides and Eighteen potential antigenic peptides at average propensity 1.063 having highest local hydrophilicity. Thus a small antigen fragment can induce immune response against whole antigen. This approach can be applied for designing subunit and synthetic peptide vaccines.

Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

DOE PAGES

Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; ...

2013-10-07

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

Multiple Antigen Peptide Vaccines against Plasmodium falciparum Malaria

DTIC Science & Technology

2010-01-01

Robert A. Boykins/ Victoria Majam,l Hong Zheng,1 Rana Chattopadhyay,l Patricia de Ia Vcga,3 J. Kathleen Moch ,J J. David Hayncs,3 Igor M. Belyakov,2...K. Moch , and D. S. Smoot. 2002. Erythroc-ytic malaria growth or invasion inhibition assays with emphasis on suspension culture GIA. Methods Mol. Med

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433

Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH.

PubMed

Calderón-González, Ricardo; Frande-Cabanes, Elisabet; Bronchalo-Vicente, Lucía; Lecea-Cuello, M Jesús; Pareja, Eduardo; Bosch-Martínez, Alexandre; Fanarraga, Mónica L; Yañez-Díaz, Sonsoles; Carrasco-Marín, Eugenio; Alvarez-Domínguez, Carmen

2014-01-01

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189-201 and LLO91-99 and the GAPDH peptide, GAPDH1-22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1-22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91-99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1-22-specific CD4(+) and CD8(+) immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4(+) and CD8(+) epitopes.

Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH

PubMed Central

Calderón-González, Ricardo; Frande-Cabanes, Elisabet; Bronchalo-Vicente, Lucía; Lecea-Cuello, M. Jesús; Pareja, Eduardo; Bosch-Martínez, Alexandre; Fanarraga, Mónica L.; Yañez-Díaz, Sonsoles; Carrasco-Marín, Eugenio; Álvarez-Domínguez, Carmen

2014-01-01

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189−201 and LLO91−99 and the GAPDH peptide, GAPDH1−22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1−22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91−99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1−22-specific CD4+ and CD8+ immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4+ and CD8+ epitopes. PMID:24600592

Anti-cancer vaccination by transdermal delivery of antigen peptide-loaded nanogels via iontophoresis.

PubMed

Toyoda, Mao; Hama, Susumu; Ikeda, Yutaka; Nagasaki, Yukio; Kogure, Kentaro

2015-04-10

Transdermal vaccination with cancer antigens is expected to become a useful anti-cancer therapy. However, it is difficult to accumulate enough antigen in the epidermis for effective exposure to Langerhans cells because of diffusion into the skin and muscle. Carriers, such as liposomes and nanoparticles, may be useful for the prevention of antigen diffusion. Iontophoresis, via application of a small electric current, is a noninvasive and efficient technology for transdermal drug delivery. Previously, we succeeded in the iontophoretic transdermal delivery of liposomes encapsulating insulin, and accumulation of polymer-based nanoparticle nanogels in the stratum corneum of the skin. Therefore, in the present study, we examined the use of iontophoresis with cancer antigen gp-100 peptide KVPRNQDWL-loaded nanogels for anti-cancer vaccination. Iontophoresis resulted in the accumulation of gp-100 peptide and nanogels in the epidermis, and subsequent increase in the number of Langerhans cells in the epidermis. Moreover, tumor growth was significantly suppressed by iontophoresis of the antigen peptide-loaded nanogels. Thus, iontophoresis of the antigen peptide-loaded nanogels may serve as an effective transdermal delivery system for anti-cancer vaccination. Copyright © 2015 Elsevier B.V. All rights reserved.

High-throughput identification and dendritic cell-based functional validation of MHC class I-restricted Mycobacterium tuberculosis epitopes

PubMed Central

Nair, Smita K.; Tomaras, Georgia D.; Sales, Ana Paula; Boczkowski, David; Chan, Cliburn; Plonk, Kelly; Cai, Yongting; Dannull, Jens; Kepler, Thomas B.; Pruitt, Scott K.; Weinhold, Kent J.

2014-01-01

Emergence of drug-resistant strains of the pathogen Mycobacterium tuberculosis (Mtb) and the ineffectiveness of BCG in curtailing Mtb infection makes vaccine development for tuberculosis an important objective. Identifying immunogenic CD8+ T cell peptide epitopes is necessary for peptide-based vaccine strategies. We present a three-tiered strategy for identifying and validating immunogenic peptides: first, identify peptides that form stable complexes with class I MHC molecules; second, determine whether cytotoxic T lymphocytes (CTLs) raised against the whole protein antigen recognize and lyse target cells pulsed with peptides that passed step 1; third, determine whether peptides that passed step 2, when administered in vivo as a vaccine in HLA-A2 transgenic mice, elicit CTLs that lyse target cells expressing the whole protein antigen. Our innovative approach uses dendritic cells transfected with Mtb antigen-encoding mRNA to drive antigen expression. Using this strategy, we have identified five novel peptide epitopes from the Mtb proteins Apa, Mtb8.4 and Mtb19. PMID:24755960

Conformational antigenic determinants generated by interactions between a bacterially expressed recombinant peptide of the hepatitis E virus structural protein.

PubMed

Zhang, J Z; Ng, M H; Xia, N S; Lau, S H; Che, X Y; Chau, T N; Lai, S T; Im, S W

2001-06-01

A 23 kDa peptide locating to amino acid residues 394 to 604 of the major Hepatitis E Virus (HEV) structural protein was expressed in E. coli. This peptide was found to interact naturally with one another to form homodimers and it was recognized strongly and commonly in its dimeric form by HEV reactive human sera. The antigenic activity associated with the dimeric form was abrogated when the dimer was dissociated into monomer and the activity was reconstituted after the monomer was re-associated into dimer again. The dimeric form of the peptide elicited a vigorous antibody response in experimental animals and the resulting antisera were found to cross-react against HEV, effecting an efficient immune capture of the virus. These results attributed the antigenic activity associated with the dimeric form of the peptide to conformational antigenic determinants generated as a result of interaction between the peptide molecules. It is suggested that some of these antigenic determinants may be expressed by the HEV capsid and raised the possibility of this bacterially expressed peptide as an HEV vaccine candidate. Copyright 2001 Wiley-Liss, Inc.

Extending antigen release from particulate vaccines results in enhanced antitumor immune response.

PubMed

Kapadia, Chintan H; Tian, Shaomin; Perry, Jillian L; Sailer, David; Christopher Luft, J; DeSimone, Joseph M

2018-01-10

Tumor-specific CD8 + cytotoxic T lymphocytes (CTLs) play a critical role in an anti-tumor immune response. However, vaccination intended to elicit a potent CD8 + T cell responses employing tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Previously, we engineered a polyethylene glycol (PEG) hydrogel-based subunit vaccine for the delivery of an antigenic peptide and CpG (adjuvant) to elicit potent CTLs. In this study, we further examined the effect of antigen release kinetics on their induced immune responses. A CD8 + T cell epitope peptide from OVA (CSIINFEKL) and CpG were co-conjugated to nanoparticles utilizing either a disulfide or a thioether linkage. Subsequent studies comparing peptide release rates as a function of linker, determined that the thioether linkage provided sustained release of peptide over 72h. Ability to control the release of peptide resulted in both higher and prolonged antigen presentation when compared to disulfide-linked peptide. Both NP vaccine formulations resulted in activation and maturation of bone marrow derived dendritic cells (BMDCs) and induced potent CD8 + T cell responses when compared to soluble antigen and soluble CpG. Immunization with either disulfide or thioether linked vaccine constructs effectively inhibited EG7-OVA tumor growth in mice, however only treatment with the thioether linked vaccine construct resulted in enhanced survival. Copyright © 2017. Published by Elsevier B.V.

Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a.

PubMed

Theillet, François-Xavier; Saul, Frederick A; Vulliez-Le Normand, Brigitte; Hoos, Sylviane; Felici, Franco; Weintraub, Andrej; Mulard, Laurence A; Phalipon, Armelle; Delepierre, Muriel; Bentley, Graham A

2009-05-15

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

PubMed

Yamamoto, A M; Cresteil, D; Boniface, O; Clerc, F F; Alvarez, F

1993-05-01

Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.

Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples *

PubMed Central

Heyder, Tina; Kohler, Maxie; Tarasova, Nataliya K.; Haag, Sabrina; Rutishauser, Dorothea; Rivera, Natalia V.; Sandin, Charlotta; Mia, Sohel; Malmström, Vivianne; Wheelock, Åsa M.; Wahlström, Jan; Holmdahl, Rikard; Eklund, Anders; Zubarev, Roman A.; Grunewald, Johan; Ytterberg, A. Jimmy

2016-01-01

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides. Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 106 bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10–15 × 106 cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls. In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 106 cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides. PMID:27452731

PS80 interferes with the antiallergic effect of Cry-consensus peptide, a novel recombinant peptide for immunotherapy of Japanese cedar pollinosis, at very low concentration through modulation of Th1/Th2 balance.

PubMed

Kozutsumi, Daisuke; Tsunematsu, Masako; Yamaji, Taketo; Murakami, Rika; Yokoyama, Minehiko; Kino, Kohsuke

2006-07-01

Polysorbate 80 (PS80 or Tween-80) is often used as an additive to promote the rapid solubilization of pharmaceuticals in aqueous solutions. We investigated whether coinjection of a minimal amount of PS80 had a modulatory effect on the immunotherapeutic effects of Cry (Cryptomeria)-consensus peptide, a novel peptide developed for the therapeutic management of Japanese cedar pollinosis, using a Cry j 1-sensitized mouse model with experimental allergic rhinitis. Subcutaneous challenge with Cry-consensus peptide plus 50 microg/ml of PS80 did not affect the antigen-specific proliferation of splenocytes, but decreased the potency of Cry-consensus peptide to inhibit antigen-specific interleukin (IL)-5 production by the cells significantly in comparison with challenge with Cry-consensus peptide alone. However, there was no significant difference between the effect of Cry-consensus peptide administration on interferon (IFN)-gamma production in the presence and absence of PS80, indicating that PS80 interfered with the T helper 1 (Th1)-dominant T helper balance induced by Cry-consensus peptide challenge. Moreover, the increase in the level of antigen-specific immunoglobulin G2a (IgG2a) induced by Cry-consensus peptide challenge was inhibited slightly but unambiguously by PS80 coinjection. These in vitro experiments indicated that PS80 induces Th2-type differentiation of T helper cells through preferential inhibition of IFN-gamma expression relative to IL-5 expression in splenocytes in a concentration-dependent manner. In naïve mice, sensitization by Cry-consensus peptide with PS80 induced antigen-specific IL-5 production more potently than sensitization by Cry-consensus peptide alone, and when PS80 was added to bone marrow-derived dendritic cells, the endocytosis of fluorescence-labelled Cry-consensus peptide was dramatically inhibited in a concentration-dependent manner. Therefore, we conclude that PS80 has an immunomodulatory effect on the antigen-specific response resulting in a shift towards Th2 predominance with respect to the antigen recognition stage. Taken together, our findings suggest that PS80 might decrease the efficacy of Cry-consensus peptide through modulation of the efficiency of antigen endocytosis and/or of the direction of successive T helper cell differentiation.

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β-turns in peptide immunogenicity.

PubMed

Shukla, Rashmi Tambe; Sasidhar, Yellamraju U

2015-07-01

Earlier immunological experiments with a synthetic 36-residue peptide (75-110) from Influenza hemagglutinin have been shown to elicit anti-peptide antibodies (Ab) which could cross-react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide ((98)YPYDVPDYASLRS(110)) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS-AA/L all-atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β-turn formed by "DYAS" sequence) with greater preference at 310 K while, it samples a native-like conformation with relatively greater propensity at 293 K. The sequence "DYAS" samples β-turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β-turn involving "DYAS" sequence and in addition it also samples a β-turn formed by the sequence "YPYD" at its N-terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag-Ab recognition are discussed. © 2015 Wiley Periodicals, Inc.

Autoantigen cross-reactive environmental antigen can trigger multiple sclerosis-like disease.

PubMed

Reynolds, Catherine J; Sim, Malcolm J W; Quigley, Kathryn J; Altmann, Daniel M; Boyton, Rosemary J

2015-05-13

Multiple sclerosis is generally considered an autoimmune disease resulting from interaction between predisposing genes and environmental factors, together allowing immunological self-tolerance to be compromised. The precise nature of the environmental inputs has been elusive, infectious agents having received considerable attention. A recent study generated an algorithm predicting naturally occurring T cell receptor (TCR) ligands from the proteome database. Taking the example of a multiple sclerosis patient-derived anti-myelin TCR, the study identified a number of stimulatory, cross-reactive peptide sequences from environmental and human antigens. Having previously generated a spontaneous multiple sclerosis (MS) model through expression of this TCR, we asked whether any of these could indeed function in vivo to trigger CNS disease by cross-reactive activation. A number of myelin epitope cross-reactive epitopes could stimulate T cell immunity in this MS anti-myelin TCR transgenic model. Two of the most stimulatory of these 'environmental' epitopes, from Dictyostyelium slime mold and from Emiliania huxleyi, were tested for the ability to induce MS-like disease in the transgenics. We found that immunization with cross-reactive peptide from Dictyostyelium slime mold (but not from E. huxleyi) induces severe disease. These specific environmental epitopes are unlikely to be common triggers of MS, but this study suggests that our search for the cross-reactivity triggers of autoimmune activation leading to MS should encompass epitopes not just from the 'infectome' but also from the full environmental 'exposome.'

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

PubMed

Spierings, Eric; Brickner, Anthony G; Caldwell, Jennifer A; Zegveld, Suzanne; Tatsis, Nia; Blokland, Els; Pool, Jos; Pierce, Richard A; Mollah, Sahana; Shabanowitz, Jeffrey; Eisenlohr, Laurence C; van Veelen, Peter; Ossendorp, Ferry; Hunt, Donald F; Goulmy, Els; Engelhard, Victor H

2003-07-15

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

PubMed

Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Induction of peripheral T cell tolerance and allo/xenoimmunity.

PubMed

Charpentier, B; Alard, P; Hiesse, C; Lantz, O

1994-03-01

It is theoretically impossible or at least difficult to tolerize animals in xenogeneic situation, particularly in non-concordant species. Both humoral defense and several cellular components (T and non T) are offensive weapons which can be directed at myriad of self-antigens in a normal situation, at allo-antigens in abnormal situations, at xeno-antigens in exceptional situations. In either cases, the fine epitopic definition of allo/xeno antigens seen by the TCR is far from being totally known. From recent studies showing the precise requirement of peptides assembly composition in the MHC class I and II groove it should be theoretically possible to tolerize any group of peptides if they are presented, some other, because not presented, may remain ignored, thus apparently tolerized. It is also know, that transplantation tolerance is not a law of "either nothing or all" but a multiple process depending of both the affinity of the TCR and the nature/compositions of the target. In view of the complex array of factors influencing the pathway of T cell activation, three forms of T cell non responsiveness may be suggested in the context of xenogeneic recognition: physical deletion of potentially reactive T cells occurring predominantly in the thymus, non reactivity of T cells resulting from their failure to be influenced by antigens, and anergy possibly due to inappropriate signals from non professional antigen presenting cells. Future investigations must elucidate the requirements for inducing these events in a purpose of xenogeneic organ transplantation.

Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

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

Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.

Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of keymore » binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.« less

Synthetic peptides for the immunodiagnosis of hepatitis A virus infection.

PubMed

Gauna, A; Losada, S; Lorenzo, M; Bermúdez, H; Toledo, M; Pérez, H; Chacón, E; Noya, O

2015-12-01

VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T-cell recognition

PubMed Central

God, Jason M; Zhao, Dan; Cameron, Christine A; Amria, Shereen; Bethard, Jennifer R; Haque, Azizul

2014-01-01

While Burkitt lymphoma (BL) has a well-known defect in HLA class I-mediated antigen presentation, the exact role of BL-associated HLA class II in generating a poor CD4+ T-cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T-cell recognition was not associated with low levels of co-stimulatory molecules on BL cells, as addition of external co-stimulation failed to elicit CD4+ T-cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II-mediated antigen presentation and CD4+ T-cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase-like molecule that enhances class II-mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II-mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies. PMID:24628049

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

PubMed Central

Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

2015-01-01

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach. PMID:25512614

Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

PubMed

Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2017-11-01

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

PubMed Central

List, Claudia; Qi, Weihong; Maag, Eva; Gottstein, Bruno; Müller, Norbert; Felger, Ingrid

2010-01-01

Background Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. Methodology/Principal Findings Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. Conclusions/Significance This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens. PMID:20689813

Design of Peptide Immunotherapies for MHC Class-II-Associated Autoimmune Disorders

PubMed Central

2013-01-01

Autoimmune disorders, that occur when autoreactive immune cells are induced to activate their responses against self-tissues, affect one percent of the world population and represent one of the top 10 leading causes of death. The major histocompatibility complex (MHC) is a principal susceptibility locus for many human autoimmune diseases, in which self-tissue antigens providing targets for pathogenic lymphocytes are bound to HLA molecules encoded by disease-associated alleles. In spite of the attempts to design strategies for inhibition of antigen presentation targeting the MHC-peptide/TCR complex via generation of blocking antibodies, altered peptide ligands (APL), or inhibitors of costimulatory molecules, potent therapies with minimal side effects have yet to be developed. Copaxone (glatiramer acetate, GA) is a random synthetic amino acid copolymer that reduces the relapse rate by about 30% in relapsing-remitting multiple sclerosis (MS) patients. Based on the elucidated binding motifs of Copaxone and of the anchor residues of the immunogenic myelin basic protein (MBP) peptide to HLA-DR molecules, novel copolymers have been designed and proved to be more effective in suppressing MS-like disease in mice. In this report, we describe the rationale for design of second-generation synthetic random copolymers as candidate drugs for a number of MHC class-II-associated autoimmune disorders. PMID:24324511

Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

PubMed Central

Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

2016-01-01

Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

Gradated assembly of multiple proteins into supramolecular nanomaterials

NASA Astrophysics Data System (ADS)

Hudalla, Gregory A.; Sun, Tao; Gasiorowski, Joshua Z.; Han, Huifang; Tian, Ye F.; Chong, Anita S.; Collier, Joel H.

2014-08-01

Biomaterials exhibiting precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibres and gels with exceptional compositional control. The strategy employs ‘βTail’ tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional β-sheet fibrillizing peptides. Multiple different βTail fusion proteins could be inserted into peptide nanofibres alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibres bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

PubMed Central

Cram, Erik D.; Simmons, Ryan S.; Palmer, Amy L.; Hildebrand, William H.; Rockey, Daniel D.

2015-01-01

The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8+ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8+ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8+ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. PMID:26597986

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

PubMed

Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

1995-01-01

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

PubMed

Dagenais, P; Desprez, B; Albert, J; Escher, E

1994-10-01

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis.

PubMed

Trier, Nicole Hartwig; Holm, Bettina Eide; Heiden, Julie; Slot, Ole; Locht, Henning; Lindegaard, Hanne; Svendsen, Anders; Nielsen, Christoffer Tandrup; Jacobsen, Søren; Theander, Elke; Houen, Gunnar

2018-02-27

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Anti-citrullinated protein antibodies (ACPA) are crucial for the serological diagnosis of RA, where Epstein-Barr virus (EBV) has been suggested to be an environmental agent in triggering the onset of the disease. This study aimed to analyse antibody reactivity to citrullinated EBV nuclear antigen-2 (EBNA-2) peptides from three different EBV strains (B95-8, GD1 and AG876) using streptavidin capture enzyme-linked immunosorbent assay. One peptide, only found in a single strain (AG876), obtained a sensitivity and specificity of 77% and 95%, respectively and showed high sequence similarity to the filaggrin peptide originally used for ACPA detection. Comparison of antibody reactivity to commercial assays found that the citrullinated peptide was as effective in detecting ACPA as highly sensitive and specific commercial assays. The data presented demonstrate that the citrullinated EBNA-2 peptide indeed is recognised specifically by RA sera and that the single peptide is able to compete with assays containing multiple peptides. Furthermore, it could be hypothesized that RA may be caused by (a) specific strain(s) of EBV.

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

PubMed Central

Lavenant, Gwendoline Thiery; Zavalin, Andrey I.; Caprioli, Richard M.

2013-01-01

Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet. PMID:23397138

Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

NASA Astrophysics Data System (ADS)

Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

2013-04-01

Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

1995-01-01

Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules*

PubMed Central

Barroso, Margarida; Tucker, Heidi; Drake, Lisa; Nichol, Kathleen; Drake, James R.

2015-01-01

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen. PMID:26400081

Peptide-loaded Langerhans cells, despite increased IL15 secretion and T cell activation in vitro, elicit anti-tumor T cell responses comparable to peptide-loaded monocyte-derived dendritic cells in vivo

PubMed Central

Romano, Emanuela; Rossi, Marco; Ratzinger, Gudrun; de Cos, Maria-Angeles; Chung, David J.; Panageas, Katherine S.; Wolchok, Jedd D.; Houghton, Alan N.; Chapman, Paul B.; Heller, Glenn; Yuan, Jianda; Young, James W.

2013-01-01

Purpose We compared the efficacy of human Langerhans cells (LCs) as tumor immunogens in vivo with monocyte-derived DCs (moDCs) and investigated how IL15 supports optimal DC-stimulated antitumor immunity. Experimental Design AJCC stage III/IV melanoma patients participated in this first clinical trial comparing melanoma peptide-pulsed LC with moDC vaccines (NCT00700167,www.ClinicalTrials.gov). Correlative studies evaluated mechanisms mediating IL15 support of DC-stimulated antitumor immunity. Results Both DC vaccines were safe and immunogenic for melanoma antigens. LC-based vaccines stimulated significantly greater tyrosinase-HLA-A*0201 tetramer reactivity than did moDC-based vaccines. The two DC subtypes were otherwise statistically comparable, in contrast to extensive prior data in vitro demonstrating LC superiority. LCs synthesize much more IL15 than moDCs and stimulate significantly more antigen-specific lymphocytes with a cytolytic IFN-gamma profile even without exogenous IL15. When supplemented by low dose IL15, instead of IL2, moDCs stimulate 5-6 logs more tumor antigen-specific effector memory T-cells (TEMRA) over 3-4 weeks in vitro. IL2 and IL15 can be synergistic in moDC stimulation of cytolytic T-cells. IL15 promotes T-cell expression of the antiapoptotic bcl-2 and inhibits candidate regulatory T-cell (Treg) expansion after DC stimulation, countering two effects of IL2 that do not foster tumor immunity. Conclusions MoDC-based vaccines will require exogenous IL15 to achieve clinical efficacy. Alternatively, LCs can couple the endogenous production of IL15 with potent T-cell stimulatory activity. Optimization of full length tumor antigen expression for processing into multiple immunogenic peptides for presentation by both class I and II MHC therefore merits emphasis to support more effective antitumor immunity stimulated by LCs. PMID:21355077

Vaccination of High-Risk Breast Cancer Patients with Carbohydrate Mimicking Peptides

DTIC Science & Technology

2008-05-01

Spontaneous pyroglutamic acid formation for peptides starting with glutamic acid or glutamine residues is not considered an impurity. Spontaneous...Examples of tumor- associated carbohydrate antigens include the gangliosides GM2, GD2, GD3, and fucosyl GM1, Globo H, polysialic acid , STn and the...directed toward gangliosides, polysialic acid , Globo, Lewis Y (LeY), and the STn antigen. Because TACA are T-cell–independent antigens and self-antigens

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.

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

PubMed

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

2014-03-15

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.

Antigen-antibody interaction. The immunodominant region of EDP208 pili.

PubMed

Worobec, E A; Paranchych, W; Parker, J M; Taneja, A K; Hodges, R S

1985-01-25

The EDP208 pilus contains a major antigenic determinant in the N-terminal dodecapeptide, as shown by E. A. Worobec, A. K. Taneja, R. S. Hodges, and W. Paranchych ((1983) J. Bacteriol. 153, 955-961). This peptide was chemically synthesized, coupled to bovine serum albumin with N-hydroxysuccinimidyl p-azido-benzoate, and used in immunoblot and enzyme-linked immunosorbent assays to show it was capable of reacting with anti-EDP208 pilus antibodies. Antibodies raised against the synthetic peptide conjugate were also capable of reacting with whole pili in these assays. To further examine the specific residues responsible for the antigenicity of this site, several peptide analogs were chemically synthesized. The relative affinity of these peptides for anti-EDP208 pilus antibodies was determined by a competitive enzyme-linked immunosorbent assay using the Fab fragment of anti-EDP208 pilus immunoglobulin G. From these results we established that the antigenic region of this peptide was the N-terminal pentapeptide, N-acetyl-Thr-Asp-Leu-Leu-Ala, and the key residues responsible for the antibody-antigen interaction are the N-acetyl-Thr1, Leu3, and Leu4. Hydrophobic interactions involving the methyl of the acetyl group and the leucine side chains make the largest contributions to the antigen-antibody interaction, while a lesser contribution is made by the Thr1 hydroxyl. The side chains of Asp2 and Ala5 contribute only weakly to the stabilization of the antigen-antibody complex.

Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of ankylosing spondylitis reduce HLA-B27 mediated presentation of multiple antigens.

PubMed

Seregin, Sergey S; Rastall, David P W; Evnouchidou, Irini; Aylsworth, Charles F; Quiroga, Dionisia; Kamal, Ram P; Godbehere-Roosa, Sarah; Blum, Christopher F; York, Ian A; Stratikos, Efstratios; Amalfitano, Andrea

2013-12-01

Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system.

Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of Ankylosing Spondylitis reduce HLA-B27 mediated presentation of multiple antigens

PubMed Central

Seregin, Sergey S.; Rastall, David P.W.; Evnouchidou, Irini; Aylsworth, Charles F.; Quiroga, Dionisia; Kamal, Ram P.; Godbehere-Roosa, Sarah; Blum, Christopher F.; York, Ian A.; Stratikos, Efstratios; Amalfitano, Andrea

2014-01-01

Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ~0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self derived peptides by the adaptive immune system. PMID:24028501

A short peptide eluted from the H-2Kb molecule of a polyomavirus-positive tumor corresponds to polyomavirus large T antigen peptide at amino acids 578 to 585 and induces polyomavirus-specific immunity.

PubMed Central

Berke, Z; Palmer, S; Bergman, T; Wester, D; Svedmyr, J; Linder, S; Jornvall, H; Dalianis, T

1996-01-01

A short peptide in complex with the H-2Kb molecule on PyRMA, a polyomavirus transfectant of the mouse lymphoma cell line RMA, was identified as a polyomavirus tumor-specific transplantation antigen. The peptide was obtained by affinity chromatography, acidic extraction, and reverse-phase high-pressure liquid chromatography (HPLC). In one HPLC fraction, a peptide sequence in which 5 of 8 amino acids, GKxGLxxA, corresponded to residues 578 to 585 of polyomavirus large T antigen was identified. In tumor rejection assays, we therefore tested three related synthetic peptides, corresponding to the octapeptide LT 578-585, GKTGLAAA; the nonapeptide LT 578-586, GKTGLAAAL; and the decapeptide LT 578-587, GKTGLAAALI. The octapeptide was found to give the most effective immunization against the outgrowth of the polyomavirus DNA-positive PyRMA tumor. However, none of the three peptides immunized against the original polyoma-virus-negative RMA line. PMID:8627788

Competitor analogs for defined T cell antigens: peptides incorporating a putative binding motif and polyproline or polyglycine spacers.

PubMed

Maryanski, J L; Verdini, A S; Weber, P C; Salemme, F R; Corradin, G

1990-01-12

We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

Immunogenicity and in vitro Protective Efficacy of a Recombinant Multistage Plasmodium falciparum Candidate Vaccine

NASA Astrophysics Data System (ADS)

Shi, Ya Ping; Hasnain, Seyed E.; Sacci, John B.; Holloway, Brian P.; Fujioka, Hisashi; Kumar, Nirbhay; Wohlhueter, Robert; Hoffman, Stephen L.; Collins, William E.; Lal, Altaf A.

1999-02-01

Compared with a single-stage antigen-based vaccine, a multistage and multivalent Plasmodium falciparum vaccine would be more efficacious by inducing "multiple layers" of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specific P. falciparum antigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages of P. falciparum. In vitro assays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponent P. falciparum vaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.

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

Characterizing the Specificity and Co-operation of Aminopeptidases in the Cytosol and ER During MHC Class I antigen Presentation1

PubMed Central

Hearn, Arron; York, Ian A.; Bishop, Courtney; Rock, Kenneth L.

2010-01-01

Many MHC class I binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can be subsequently trimmed by aminopeptidases in the cytosol and/or the ER to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for antigen presentation is not well defined. Here we investigate this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of antigen presentation. In many cases MHC class I binding peptides are produced through sequential trimming in both the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Since N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented. PMID:20351195

Detection and Site Localization of Phosphorylcholine-Modified Peptides by NanoLC-ESI-MS/MS Using Precursor Ion Scanning and Multiple Reaction Monitoring Experiments

NASA Astrophysics Data System (ADS)

Timm, Thomas; Lenz, Christof; Merkel, Dietrich; Sadiffo, Christian; Grabitzki, Julia; Klein, Jochen; Lochnit, Guenter

2015-03-01

Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline ( m/z 104.1) and phosphorylcholine ( m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody.

PubMed

Ito, W; Nishimura, M; Sakato, N; Fujio, H; Arata, Y

1987-09-01

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.

Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

PubMed

Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

2016-01-01

This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

Parasite Manipulation of the Invariant Chain and the Peptide Editor H2-DM Affects Major Histocompatibility Complex Class II Antigen Presentation during Toxoplasma gondii Infection

PubMed Central

Nishi, Manami; El-Hage, Sandy; Fox, Barbara A.; Bzik, David J.

2015-01-01

Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4+ T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4+ T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway. PMID:26195549

Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

PubMed Central

Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

2015-01-01

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity.

PubMed

Kawa, Diane E; Stephens, Richard S

2002-05-15

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

PubMed Central

Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

2013-01-01

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

Conjugates of Cell Adhesion Peptides for Therapeutics and Diagnostics Against Cancer and Autoimmune Diseases.

PubMed

Moral, Mario E G; Siahaan, Teruna J

2017-01-01

Overexpressed cell-surface receptors are hallmarks of many disease states and are often used as markers for targeting diseased cells over healthy counterparts. Cell adhesion peptides, which are often derived from interacting regions of these receptor-ligand proteins, mimic surfaces of intact proteins and, thus, have been studied as targeting agents for various payloads to certain cell targets for cancers and autoimmune diseases. Because many cytotoxic agents in the free form are often harmful to healthy cells, the use of cell adhesion peptides in targeting their delivery to diseased cells has been studied to potentially reduce required effective doses and associated harmful side-effects. In this review, multiple cell adhesion peptides from extracellular matrix and ICAM proteins were used to selectively direct drug payloads, signal-inhibitor peptides, and diagnostic molecules, to diseased cells over normal counterparts. RGD constructs have been used to improve the selectivity and efficacy of diagnostic and drug-peptide conjugates against cancer cells. From this precedent, novel conjugates of antigenic and cell adhesion peptides, called Bifunctional Peptide Inhibitors (BPIs), have been designed to selectively regulate immune cells and suppress harmful inflammatory responses in autoimmune diseases. Similar peptide conjugations with imaging agents have delivered promising diagnostic methods in animal models of rheumatoid arthritis. BPIs have also been shown to generate immune tolerance and suppress autoimmune diseases in animal models of type-1 diabetes, rheumatoid arthritis, and multiple sclerosis. Collectively, these studies show the potential of cell adhesion peptides in improving the delivery of drugs and diagnostic agents to diseased cells in clinical settings. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

PubMed

Chuan, Yap P; Wibowo, Nani; Connors, Natalie K; Wu, Yang; Hughes, Fiona K; Batzloff, Michael R; Lua, Linda H L; Middelberg, Anton P J

2014-06-01

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 10(4) ). Second, delivery by capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 μg (corresponding to <600 ng of each GAS peptide), induced significant titers of IgGs specific to each peptide, demonstrating that a multivalent, broad-coverage VLP vaccine formulation was possible. In summary, the modular VLPs and capsomeres reported here demonstrate, with promising preliminary data, innovative ways to design GAS vaccines using VLP and capsomere delivery systems amenable to microbial synthesis, potentially adoptable by developing countries. © 2013 Wiley Periodicals, Inc.

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition*

PubMed Central

Hawse, William F.; Gloor, Brian E.; Ayres, Cory M.; Kho, Kevin; Nuter, Elizabeth; Baker, Brian M.

2013-01-01

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity. PMID:23836912

Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

PubMed Central

Schiavone, Marco; Fiume, Giuseppe; Caivano, Antonella; de Laurentiis, Annamaria; Falcone, Cristina; Masci, Francesca Fasanella; Iaccino, Enrico; Mimmi, Selena; Palmieri, Camillo; Pisano, Antonio; Pontoriero, Marilena; Rossi, Annalisa; Scialdone, Annarita; Vecchio, Eleonora; Andreozzi, Concetta; Trovato, Maria; Rafay, Jan; Ferko, Boris; Montefiori, David; Lombardi, Angela; Morsica, Giulia; Poli, Guido; Quinto, Ileana; Pavone, Vincenzo; de Berardinis, Piergiuseppe; Scala, Giuseppe

2012-01-01

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV+ broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine. PMID:22754323

Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

PubMed

Yoshikawa, Toshiaki; Takahara, Masashi; Tomiyama, Mai; Nieda, Mie; Maekawa, Ryuji; Nakatsura, Tetsuya

2014-11-01

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3‑specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Humoral immunity response to human endogenous retroviruses K/W differentiates between amyotrophic lateral sclerosis and other neurological diseases.

PubMed

Arru, G; Mameli, G; Deiana, G A; Rassu, A L; Piredda, R; Sechi, E; Caggiu, E; Bo, M; Nako, E; Urso, D; Mariotto, S; Ferrari, S; Zanusso, G; Monaco, S; Sechi, G; Sechi, L A

2018-03-31

Human endogenous retroviruses (HERV) K/W seem to play a role in fostering and exacerbation of some neurological diseases, including amyotrophic lateral sclerosis (ALS). Given these findings, the immunity response against HERV-K and HERV-W envelope surface (env-su) glycoprotein antigens in serum and cerebrospinal fluid (CSF) was investigated for ALS, multiple sclerosis (MS) and Alzheimer's disease patients and in healthy controls. Four antigenic peptides derived respectively from HERV-K and HERV-W env-su proteins were studied in 21 definite or probable ALS patients, 26 possible or definite relapsing-remitting MS patients, 18 patients with Alzheimer's disease and 39 healthy controls. An indirect enzyme-linked immunosorbent assay was set up to detect specific antibodies (Abs) against env-su peptides. Amongst the measured levels of Abs against the four different HERV-K peptide fragments, only HERV-K env-su 19-37 was significantly elevated in ALS compared to other groups, both in serum and CSF. Instead, amongst the Abs levels directed against the four different HERV-W peptide fragments, only HERV-W env-su 93-108 and HERV-W env-su 248-262 were significantly elevated, in the serum and CSF of the MS group compared to other groups. In ALS patients, the HERV-K env-su 19-37 Abs levels were significantly correlated with clinical measures of disease severity, both in serum and CSF. Increased circulating levels of Abs directed against the HERV-W env-su 93-108 and HERV-W env-su 248-262 peptide fragments could serve as possible biomarkers in patients with MS. Similarly, increased circulating levels of Abs directed against the HERV-K env-su 19-37 peptide fragment could serve as a possible early novel biomarker in patients with ALS. © 2018 EAN.

Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques

PubMed Central

Li, Hongzhao; Nykoluk, Mikaela; Li, Lin; Liu, Lewis R.; Omange, Robert W.; Soule, Geoff; Schroeder, Lukas T.; Toledo, Nikki; Kashem, Mohammad Abul; Correia-Pinto, Jorge F.; Liang, Binhua; Schultz-Darken, Nancy; Alonso, Maria J.; Whitney, James B.; Plummer, Francis A.

2017-01-01

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research. PMID:28982126

The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes*

PubMed Central

Hassan, Chopie; Kester, Michel G. D.; de Ru, Arnoud H.; Hombrink, Pleun; Drijfhout, Jan Wouter; Nijveen, Harm; Leunissen, Jack A. M.; Heemskerk, Mirjam H. M.; Falkenburg, J. H. Frederik; van Veelen, Peter A.

2013-01-01

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome. PMID:23481700

Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

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

T Nguyen; S Chang; I Evnouchidou

2011-12-31

ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of longmore » rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.« less

Characteristics of the pre-diabetic period in children with high risk of type 1 diabetes recruited from the general Swedish population-The ABIS study.

PubMed

Åkerman, Linda; Ludvigsson, Johnny; Swartling, Ulrica; Casas, Rosaura

2017-09-01

There is a need for increased understanding of the pre-diabetic period in individuals with high risk of type 1 diabetes from the general population. High-risk children (n = 21) positive for multiple islet autoantibodies were identified by autoantibody screening within the All Babies in Southeast Sweden study. The children and their parents were enrolled in a 2-year prospective follow-up study aiming to characterize the pre-diabetic period. Blood samples were collected every 6 months for measurement of C-peptide, HbA1c, fasting glucose, and autoantibodies. Human leukocyte antigen-genotype was determined, and oral glucose tolerance test was performed every 12 months. Despite positivity for multiple autoantibodies, 9 out of 21 individuals had low-risk human leukocyte antigen-genotypes. Children who progressed to manifest diabetes (progressors, n = 12) had higher levels of IA2A and ZnT8A than children who did not (non-progressors, n = 9). Impaired glucose tolerance and impaired fasting glucose was observed to the same extent in progressors and non-progressors, but HbA1c increased over time in progressors in spite of increased C-peptide. Autoantibodies to IA2 and ZnT8 may be useful discriminators for disease progression in at-risk children from the general population. Dysglycemia was observed long before diagnosis, and difficulties in maintaining glucose homeostasis despite increased C-peptide indicate that insulin resistance might be an important accelerator of disease in risk individuals. Copyright © 2017 John Wiley & Sons, Ltd.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

PubMed Central

Boisgerault, F; Khalil, I; Tieng, V; Connan, F; Tabary, T; Cohen, J H; Choppin, J; Charron, D; Toubert, A

1996-01-01

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy. PMID:8622959

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles.

PubMed Central

Nisini, R; Paroli, M; Accapezzato, D; Bonino, F; Rosina, F; Santantonio, T; Sallusto, F; Amoroso, A; Houghton, M; Barnaba, V

1997-01-01

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection. PMID:9032359

Targeting nanosystems to human DCs via Fc receptor as an effective strategy to deliver antigen for immunotherapy.

PubMed

Cruz, Luis J; Rueda, Felix; Cordobilla, Begoña; Simón, Lorena; Hosta, Leticia; Albericio, Fernando; Domingo, Joan Carles

2011-02-07

Dendritic cells (DCs) are increasingly being explored as cellular vaccines for tumor immunotherapy, since they provide an effective system of antigen presentation both in vitro and in vivo. An additional advantage of this cell type is that it is possible to target specific antigens through the activation of receptors, such as FcR (the receptor for the IgG Fc fragment) and TLR (toll-like Receptor). Thus, the uptake capacity of DCs can be improved, thereby increasing antigen presentation. This, in turn, would lead to an enhanced immune response, and, in some instances, the tolerance/anergy of immune effector cells present in cancer patients could be reverted. Here we studied various nanotargeting systems, including liposomes and gold nanoparticles of a peptide-based immunotherapeutic vaccine for the treatment of androgen-responsive prostate cancer. Building blocks of the immunogenic peptide consisted of the luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH) peptide (B- and T-cell epitope), in tandem with a T-helper epitope corresponding to the 830-844 region of tetanus toxoid. Three new peptides with several modifications at the N-terminal (palmitoyl, acetyl, and FITC) were synthesized. These peptides also contained a Cys as C-terminal residue to facilitate grafting onto gold nanoparticles. To target different antigen formulations to human DCs, the Fc was activated with a cross-linking spacer to generate a free thiol group and thus facilitate conjugation onto gold nanoparticles, liposomes, and peptide. Our results show that gold nanoparticles and liposomes targeted to FcRs of human DCs are effective antigen delivery carriers and induce a strong immune response with respect to nontargeted LHRH-TT-nanoparticle conjugates and a superior response to that of naked antigens. In addition, dual labeling using gold and FITC-peptide allowed DC tracking by flow cytometry as well as transmission electron microscopy. Nanoparticles were observed to show a homogeneous distribution throughout the cytoplasm. These results open up a new approach to the development of a novel strategy for cancer vaccines.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs

PubMed Central

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A.; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J.; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P.; Laclette, Juan P.

2017-01-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants. PMID:28945737

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

PubMed

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P

2017-09-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

Improvement of Peptide-Based Tumor Immunotherapy Using pH-Sensitive Fusogenic Polymer-Modified Liposomes.

PubMed

Yoshizaki, Yuta; Yuba, Eiji; Komatsu, Toshihiro; Udaka, Keiko; Harada, Atsushi; Kono, Kenji

2016-09-26

To establish peptide vaccine-based cancer immunotherapy, we investigated the improvement of antigenic peptides by encapsulation with pH-sensitive fusogenic polymer-modified liposomes for induction of antigen-specific immunity. The liposomes were prepared by modification of egg yolk phosphatidylcholine and l-dioleoyl phosphatidylethanolamine with 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG) and were loaded with antigenic peptides derived from ovalbumin (OVA) OVA-I (SIINFEKL), and OVA-II (PSISQAVHAAHAEINEAP β A), which bind, respectively, to major histocompatibility complex (MHC) class I and class II molecules on dendritic cell (DCs). The peptide-loaded liposomes were taken up efficiently by DCs. The peptides were delivered into their cytosol. Administration of OVA-I-loaded MGlu-HPG-modified liposomes to mice bearing OVA-expressing E.G7-OVA tumors induced the activation of OVA-specific CTLs much more efficiently than the administration of free OVA-I peptide did. Mice strongly rejected E.G7-OVA cells after immunization with OVA-I peptide-loaded MGlu-HPG liposomes, although mice treated with free OVA-I peptide only slightly rejected the cells. Furthermore, efficient suppression of tumor volume was observed when tumor-bearing mice were immunized with OVA-I-peptide-loaded liposomes. Immunization with OVA-II-loaded MGlu-HPG-modified liposomes exhibited much lower tumor-suppressive effects. Results indicate that MGlu-HPG liposomes might be useful for improvement of CTL-inducing peptides for efficient cancer immunotherapy.

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

PubMed

Kakimi, Kazuhiro; Isobe, Midori; Uenaka, Akiko; Wada, Hisashi; Sato, Eiichi; Doki, Yuichiro; Nakajima, Jun; Seto, Yasuyuki; Yamatsuji, Tomoki; Naomoto, Yoshio; Shiraishi, Kenshiro; Takigawa, Nagio; Kiura, Katsuyuki; Tsuji, Kazuhide; Iwatsuki, Keiji; Oka, Mikio; Pan, Linda; Hoffman, Eric W; Old, Lloyd J; Nakayama, Eiichi

2011-12-15

We conducted a phase I clinical trial of a cancer vaccine using a 20-mer NY-ESO-1f peptide (NY-ESO-1 91-110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 μg of NY-ESO-1f peptide mixed with 0.2 KE Picibanil OK-432 and 1.25 ml Montanide ISA-51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY-ESO-1f peptide vaccine was well tolerated. Vaccine-related adverse events observed were fever (Grade 1), injection-site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY-ESO-1f peptide resulted in an increase or induction of NY-ESO-1 antibody responses in nine of ten patients. The sera reacted with recombinant NY-ESO-1 whole protein as well as the NY-ESO-1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine-induced CD4 and CD8 T cells responded to NY-ESO-1 91-108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20-mer NY-ESO-1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY-ESO-1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients. Copyright © 2011 UICC.

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.

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

PubMed Central

Simmons, Daniel T.; Martin, Malcolm A.; Mora, Peter T.; Chang, Chungming

1980-01-01

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences. Images PMID:6247503

The impact of human leukocyte antigen (HLA) micropolymorphism on ligand specificity within the HLA-B*41 allotypic family

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

Bade-Döding, Christina; Theodossis, Alex; Gras, Stephanie

2011-09-28

Polymorphic differences between human leukocyte antigen (HLA) molecules affect the specificity and conformation of their bound peptides and lead to differential selection of the T-cell repertoire. Mismatching during allogeneic transplantation can, therefore, lead to immunological reactions. We investigated the structure-function relationships of six members of the HLA-B*41 allelic group that differ by six polymorphic amino acids, including positions 80, 95, 97 and 114 within the antigen-binding cleft. Peptide-binding motifs for B*41:01, *41:02, *41:03, *41:04, *41:05 and *41:06 were determined by sequencing self-peptides from recombinant B*41 molecules by electrospray ionization tandem mass spectrometry. The crystal structures of HLA-B*41:03 bound to amore » natural 16-mer self-ligand (AEMYGSVTEHPSPSPL) and HLA-B*41:04 bound to a natural 11-mer self-ligand (HEEAVSVDRVL) were solved. Peptide analysis revealed that all B*41 alleles have an identical anchor motif at peptide position 2 (glutamic acid), but differ in their choice of C-terminal p{Omega} anchor (proline, valine, leucine). Additionally, B*41:04 displayed a greater preference for long peptides (>10 residues) when compared to the other B*41 allomorphs, while the longest peptide to be eluted from the allelic group (a 16mer) was obtained from B*41:03. The crystal structures of HLA-B*41:03 and HLA-B*41:04 revealed that both alleles interact in a highly conserved manner with the terminal regions of their respective ligands, while micropolymorphism-induced changes in the steric and electrostatic properties of the antigen-binding cleft account for differences in peptide repertoire and auxiliary anchoring. Differences in peptide repertoire, and peptide length specificity reflect the significant functional evolution of these closely related allotypes and signal their importance in allogeneic transplantation, especially B*41:03 and B*41:04, which accommodate longer peptides, creating structurally distinct peptide-HLA complexes.« less

Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

PubMed

Liu, Xiao-Qi; Jiang, Rong; Li, Si-Qi; Wang, Jing; Yi, Fa-Ping

2015-01-01

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

PubMed Central

Thiel, U; Pirson, S; Müller-Spahn, C; Conrad, H; Busch, D H; Bernhard, H; Burdach, S; Richter, G H S

2011-01-01

Background: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8+ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. Methods: Following repetitive CHM1319 (VIMPCSWWV) and EZH2666 (YMCSFLFNL) peptide-driven stimulations with HLA-A*0201+ dendritic cells (DC), allo-restricted HLA-A*0201− CD8+ T cells were stained with HLA-A*0201/peptide multimers, sorted and expanded by limiting dilution. Results: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A*0201 and killed HLA-A*0201+ ET lines expressing the antigen while HLA-A*0201– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2−/−γC−/− mice. Within this context, we identified the CHM1319 peptide as a new candidate target antigen for ET immunotherapy. Conclusion: These results clearly identify the ET-derived antigens, EZH2666 and CHM1319, as suitable targets for protective allo-restricted human CD8+ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation. PMID:21407224

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells

PubMed Central

Peters, Haley L.; Tripathi, Satyendra C.; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R.; St. John, Lisa S.; Federico, Lorenzo; Meraz, Ismail M.; Roth, Jack A.; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L.; Heymach, John V.; Swisher, Stephen G.; Bernantchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J.

2017-01-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these re-invigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAM were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTL from healthy donors that had been expanded against select peptides. Finally, CTL specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. PMID:28254787

Localization of sequential antigenic determinants on bovine beta-casein with synthetic peptides and antisera from mouse, rabbit, and goat.

PubMed

Mizumachi, K; Kurisaki, J; Kaminogawa, S

1999-05-01

The antigenic determinants of bovine beta-casein (beta-CN) were localized by using twenty overlapping peptides encompassing the entire sequence of beta-CN and anti-beta-CN antisera from outbred mouse, rabbit and goat. The profile of the reactions was characteristic to the species, the dominant antigenic regions being 80-95, 143-158 and 195-209 in mouse, 1-16 in rabbit and 100-115 in goat. Regions 1-16, 100-115, 121-136 and 143-158 were antigenic in all three species. The number of antigenic regions recognized by goat was much fewer than that by mouse and rabbit, possibly because of the homology between bovine and goat beta-CN. A mixture of the twenty peptides could absorb about 50-60% of beta-CN specific antibodies from each species. Furthermore, the mouse and rabbit anti-beta-CN antibodies were also specific to the phosphorylated regions. We therefore conclude that the major antigenic determinants on beta-CN would be largely sequential and include the phosphorylated sites.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants*

PubMed Central

Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán

2015-01-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

PubMed

Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

2015-07-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Polyomavirus BK Large T-Antigen-Derived Peptide Elicits an HLA-DR Promiscuous and Polyfunctional CD4+ T-Cell Response▿

PubMed Central

Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S.

2011-01-01

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4+ T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4+ T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols. PMID:21367979

The polyomavirus BK large T-antigen-derived peptide elicits an HLA-DR promiscuous and polyfunctional CD4+ T-cell response.

PubMed

Ramaswami, Bala; Popescu, Iulia; Macedo, Camila; Luo, Chunqing; Shapiro, Ron; Metes, Diana; Chalasani, Geetha; Randhawa, Parmjeet S

2011-05-01

BK virus (BKV) nephropathy and hemorrhagic cystitis are increasingly recognized causes of disease in renal and hematopoietic stem cell transplant recipients, respectively. Functional characterization of the immune response to BKV is important for clinical diagnosis, prognosis, and vaccine design. A peptide mix (PepMix) and overlapping (OPP) or random (RPP) peptide pools derived from BKV large T antigen (LTA) were used to restimulate 14-day-expanded peripheral blood mononuclear cells (PBMC) from 27 healthy control subjects in gamma interferon (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assays. A T-cell response to LTA PepMix was detected in 15/27 subjects. A response was frequently observed with peptides derived from the helicase domain (9/15 subjects), while the DNA binding and host range domains were immunologically inert (0/15 subjects). For all nine subjects who responded to LTA peptide pools, the immune response could be explained largely by a 15-mer peptide designated P313. P313-specific CD4(+) T-cell clones demonstrated (i) stringent LTA peptide specificity; (ii) promiscuous recognition in the context of HLA-DR alleles; (iii) cross recognition of homologous peptides from the polyomavirus simian virus 40 (SV40); (iv) an effector memory phenotype, CD107a expression, and intracellular production of IFN-γ and tumor necrosis factor alpha (TNF-α); (v) cytotoxic activity in a chromium release assay; and (vi) the ability to directly present cognate antigen to autologous T cells. In conclusion, T-cell-mediated immunity to BKV in healthy subjects is associated with a polyfunctional population of CD4(+) T cells with dual T-helper and T-cytotoxic properties. HLA class II promiscuity in antigen presentation makes the targeted LTA peptide sequence a suitable candidate for inclusion in immunotherapy protocols.

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

PubMed

Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

2015-10-23

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

PubMed

Weber, Laura K; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen; Dübel, Stefan; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F

2017-04-01

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Active immunization with the peptide epitope vaccine Aβ3-10-KLH induces a Th2-polarized anti-Aβ antibody response and decreases amyloid plaques in APP/PS1 transgenic mice.

PubMed

Ding, Li; Meng, Yuan; Zhang, Hui-Yi; Yin, Wen-Chao; Yan, Yi; Cao, Yun-Peng

2016-11-10

Active amyloid-β (Aβ) immunotherapy is effective in preventing Aβ deposition, facilitating plaque clearance, and improving cognitive functions in mouse models of Alzheimer's disease (AD). Developing a safe and effective AD vaccine requires a delicate balance between inducing adequate humoral immune responses and avoiding T cell-mediated autoimmune responses. In this study, we designed 2 peptide epitope vaccines, Aβ3-10-KLH and 5Aβ3-10, prepared respectively by coupling Aβ3-10 to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) or by joining 5 Aβ3-10 epitopes linearly in tandem. Young APP/PS1 mice were immunized subcutaneously with Aβ3-10-KLH or 5Aβ3-10 mixed with Freund's adjuvant, and the immunopotencies of these Aβ3-10 peptide vaccines were tested. Aβ3-10-KLH elicited a robust Th2-polarized anti-Aβ antibody response and inhibited Aβ deposition in APP/PS1 mice. However, 5Aβ3-10 did not induce an effective humoral immune response. These results indicated that Aβ3-10-KLH may be a safe and efficient vaccine for AD and that conjugating the antigen to a carrier protein may be more effective than linking multiple peptide antigens in tandem in applications for antibody production and vaccine preparation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Computational design of d-peptide inhibitors of hepatitis delta antigen dimerization

NASA Astrophysics Data System (ADS)

Elkin, Carl D.; Zuccola, Harmon J.; Hogle, James M.; Joseph-McCarthy, Diane

2000-11-01

Hepatitis delta virus (HDV) encodes a single polypeptide called hepatitis delta antigen (DAg). Dimerization of DAg is required for viral replication. The structure of the dimerization region, residues 12 to 60, consists of an anti-parallel coiled coil [Zuccola et al., Structure, 6 (1998) 821]. Multiple Copy Simultaneous Searches (MCSS) of the hydrophobic core region formed by the bend in the helix of one monomer of this structure were carried out for many diverse functional groups. Six critical interaction sites were identified. The Protein Data Bank was searched for backbone templates to use in the subsequent design process by matching to these sites. A 14 residue helix expected to bind to the d-isomer of the target structure was selected as the template. Over 200 000 mutant sequences of this peptide were generated based on the MCSS results. A secondary structure prediction algorithm was used to screen all sequences, and in general only those that were predicted to be highly helical were retained. Approximately 100 of these 14-mers were model built as d-peptides and docked with the l-isomer of the target monomer. Based on calculated interaction energies, predicted helicity, and intrahelical salt bridge patterns, a small number of peptides were selected as the most promising candidates. The ligand design approach presented here is the computational analogue of mirror image phage display. The results have been used to characterize the interactions responsible for formation of this model anti-parallel coiled coil and to suggest potential ligands to disrupt it.

Epitopes of Microbial and Human Heat Shock Protein 60 and Their Recognition in Myalgic Encephalomyelitis

PubMed Central

Elfaitouri, Amal; Herrmann, Björn; Bölin-Wiener, Agnes; Wang, Yilin; Gottfries, Carl-Gerhard; Zachrisson, Olof; Pipkorn, Rϋdiger; Rönnblom, Lars; Blomberg, Jonas

2013-01-01

Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity. PMID:24312270

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

A systems approach to designing next generation vaccines: combining α-galactose modified antigens with nanoparticle platforms

NASA Astrophysics Data System (ADS)

Phanse, Yashdeep; Carrillo-Conde, Brenda R.; Ramer-Tait, Amanda E.; Broderick, Scott; Kong, Chang Sun; Rajan, Krishna; Flick, Ramon; Mandell, Robert B.; Narasimhan, Balaji; Wannemuehler, Michael J.

2014-01-01

Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4+ T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.

Characterization of MHC-II antigen presentation by B cells and monocytes from older individuals

PubMed Central

HL, Clark; R, Banks; L, Jones; TR, Hornick; PA, Higgins; CJ, Burant; DH, Canaday

2012-01-01

In this study we examine the effects of aging on antigen presentation of B cells and monocytes. We compared the antigen presentation function of peripheral blood B cells from young and old subjects using a system that specifically measures the B cell receptor (BCR)-mediated MHC-II antigen presentation. Monocytes were studied as well. Overall the mean magnitude of antigen presentation of soluble antigen and peptide was not different in older and younger subjects for both B cells and monocytes. Older subjects, however, showed increased heterogeneity of BCR-mediated antigen presentation by their B cells. The magnitude and variability of peptide presentation, which does not require uptake and processing, was the same between groups. Presentation by monocytes had similar variability between the older and younger subjects. These data suggest that poor B cell antigen processing, which results in diminished presentation in some older individuals may contribute to poor vaccine responses. PMID:22797466

Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo1

PubMed Central

Kawahara, Masahiro; York, Ian A.; Hearn, Arron; Farfan, Diego; Rock, Kenneth L.

2015-01-01

Previous experiments using enzyme inhibitors and RNAi in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I antigen presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared to wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits antigen presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus (LCMV). The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild type and TPPII-deficient mice. These data indicate while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several antigens in vivo. PMID:19841172

Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis

PubMed Central

Yu, Hui-Chun; Lu, Ming-Chi; Li, Chin; Huang, Hsien-Lu; Huang, Kuang-Yung; Liu, Su-Qin; Lai, Ning-Sheng; Huang, Hsien-Bin

2013-01-01

The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His6-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS. PMID:24155957

Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS.

PubMed

Kruh-Garcia, Nicole A; Wolfe, Lisa M; Chaisson, Lelia H; Worodria, William O; Nahid, Payam; Schorey, Jeff S; Davis, J Lucian; Dobos, Karen M

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.

Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

PubMed Central

Kruh-Garcia, Nicole A.; Wolfe, Lisa M.; Chaisson, Lelia H.; Worodria, William O.; Nahid, Payam; Schorey, Jeff S.; Davis, J. Lucian; Dobos, Karen M.

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states. PMID:25080351

Characterization of binding specificities of Bovine Leucocyte class I molecules: Impacts for rational epitope discovery

USDA-ARS?s Scientific Manuscript database

The binding of peptides to classical major histocompatibility complex (MHC) class-I proteins is the single most selective step in antigen presentation. However, the peptide binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Her...

Poly-L-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines.

PubMed

Lingnau, Karen; Egyed, Alena; Schellack, Carola; Mattner, Frank; Buschle, Michael; Schmidt, Walter

2002-10-04

This study describes an entirely synthetic vaccine composed of antigenic peptides (T cell epitopes), oligodeoxynucleotides containing CpG-motifs (CpG-ODN) and poly-L-arginine (pR). CpG-ODN are known to be potent inducers of predominantly type 1-like immune responses, while polycationic amino acids, like pR, facilitate the uptake of antigens into antigen presenting cells (APCs). We demonstrate that the application of peptides and pR/CpG-ODN results in strongly enhanced peptide-specific immune responses as compared to the application of peptides with either of the immunomodulators alone. High numbers of antigen-specific T cells can be observed even after only one injection of the vaccine for a remarkably long period of time (at least 372 days). Furthermore, the potentially harmful systemic release of pro-inflammatory cytokines induced upon injection of CpG-ODN is inhibited. Thus, the combined application of CpG-ODN and pR may represent a novel vaccine strategy in humans.

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8+ T cell epitopes

PubMed Central

Hammond, A S; Klein, M R; Corrah, T; Fox, A; Jaye, A; McAdam, K P; Brookes, R H

2005-01-01

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8+ T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-γ. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8+ T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8+ T cell responses appear to be widespread throughout the genome. PMID:15762882

T-Cell response profiling to biological threat agents including the SARS coronavirus.

PubMed

Gioia, C; Horejsh, D; Agrati, C; Martini, F; Capobianchi, M R; Ippolito, G; Poccia, F

2005-01-01

The emergence of pathogens such as SARS and the increased threat of bioterrorism has stimulated the development of novel diagnostic assays for differential diagnosis. Rather than focusing on the detection of an individual pathogen component, we have developed a T cell profiling system to monitor responses to the pathogens in an array format. Using a matrix of antigens specific for different pathogens, a specific T cell profile was generated for each individual by monitoring the intracellular production of interferon-gamma by flow cytometry. This assay allows for the testing of multiple proteins or peptides at a single time and provides a quantitative and phenotypic assessment of CD4(+) and CD8(+) responding cells. We present profiling examples for several positive individuals, including those vaccinated with the smallpox and anthrax vaccines. We also show antigen optimization for the SARS-hCoV, as studies revealed that these proteins contain peptides which cross-react with more common coronaviruses, a cause of the common cold. The T cell array is an early and sensitive multiplex measure of active infection, exposure to a pathogen, or effective, recent vaccination.

Peptide Vaccines for Leishmaniasis.

PubMed

De Brito, Rory C F; Cardoso, Jamille M De O; Reis, Levi E S; Vieira, Joao F; Mathias, Fernando A S; Roatt, Bruno M; Aguiar-Soares, Rodrigo Dian D O; Ruiz, Jeronimo C; Resende, Daniela de M; Reis, Alexandre B

2018-01-01

Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

Sarcocystis neurona Merozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of the Toxoplasma gondii Surface Antigens (SAGs) and SAG-Related Sequences†

PubMed Central

Howe, Daniel K.; Gaji, Rajshekhar Y.; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-01-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s). PMID:15664946

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-02-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

Sheep antibodies to soluble rat collagen

PubMed Central

Chidlow, J. W.; Bourne, F. J.; Bailey, A. J.

1974-01-01

Sheep were immunized by multiple injections of acid-extracted rat tail tendon tropocollagen. Antibody activity could be demonstrated by quantitative precipitation and passive haemagglutination against denatured tropocollagen. Immunodiffusion experiments showed strong precipitin lines with denatured tendon tropocollagen, and with peptides obtained by CNBr digestion of whole rat tail tendon. Immunoelectrophoresis showed one line with denatured tropocollagen but four lines with the CNBr digest of whole tendon indicating at least four antigenic determinants. Immunosorbents prepared from antisera raised against tropocollagen readily absorbed labelled peptides from CNBr digests of rat tail tendons reduced with tritiated borohydride. These peptides were recoverable by desorption with 1 M ammonia and had a hydroxyproline and hydroxylysine content typical of collagen but increased tyrosine levels. Presence of the normal reducible components of collagen known to be involved in cross-linking was confirmed by ion-exchange chromatography, and there was an increase in the proportion of fraction C. The majority of the tritium label was found in a cross-linked peptide, or group of peptides, with molecular weight around 60,000. The technique therefore has the potential for further development in the isolation of specific collagen peptides. ImagesFIG. 3FIG. 4 PMID:4140151

Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter

PubMed Central

Kroger, Charles J; Alexander-Miller, Martha A

2007-01-01

The generation of an optimal CD8+ cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8+ cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8α and CD8β. Importantly the ensuing CD8low and CD8high CTL populations were not the result of the selective outgrowth of naive CD8+ T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to ‘fine tune’ the sensitivity of responding CTL to a defined concentration of antigen. PMID:17484768

Diagnosis of Herpes Simplex Encephalitis by ELISA Using Antipeptide Antibodies Against Type-Common Epitopes of Glycoprotein B of Herpes Simplex Viruses.

PubMed

Bhullar, Shradha S; Chandak, Nitin H; Baheti, Neeraj N; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

2016-01-01

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system (CNS). As effective antiviral drugs are available, an early, rapid, and reliable diagnosis has become important. The objective of this article was to develop a sensitive ELISA protocol for herpes simplex viruses (HSV) antigen detection and quantitation by assessing the usefulness of antipeptide antibodies against potential peptides of HSV glycoprotein B (gB). A total of 180 cerebrospinal fluid (CSF) samples of HSE and non-HSE patients were analyzed using a panel of antipeptide antibodies against synthetic peptides of HSV glycoprotein gB. The cases of confirmed and suspected HSE showed 80% and 51% positivity for antipeptide against synthetic peptide QLHDLRF and 77% and 53% positivity for antipeptide against synthetic peptide MKALYPLTT, respectively for the detection of HSV antigen in CSF. The concentration of HSV antigen was found to be higher in confirmed HSE as compared to suspected HSE group and the viral load correlated well with antigen concentration obtained using the two antipeptides in CSF of confirmed HSE group. This is the first article describing the use of antibodies obtained against synthetic peptides derived from HSV in diagnostics of HSE using patients' CSF samples.

Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes.

PubMed

Dangles, Virginie; Halberstam, Ilan; Scardino, Antonio; Choppin, Jeannine; Wertheimer, Mireille; Richon, Sophie; Quelvennec, Erwann; Moirand, Romain; Guillet, Jean-Gérard; Kosmatopoulos, Kostas; Bellet, Dominique; Zeliszewski, Dominique

2002-02-01

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.

Mucosal and systemic adjuvant effects of cholera toxin and Cry1Ac protoxin on the specific antibody response to HIV-1 C4/V3 peptides are different and depend on the antigen co-administered.

PubMed

Esquivel-Pérez, Rita; Moreno-Fierros, Leticia

2005-01-01

Evidence from several sources support the assertion that cholera toxin (CT) is a potent immunogen and mucosal adjuvant; however there are also reports showing its lack of adjuvanticity against some antigens. Cry1Ac protoxin also exerts adjuvant effects in the antibody responses to proteins and polysaccharides but its adjuvanticity with regard to peptide vaccines had not been tested. To probe whether the adjuvant effects of these proteins varied depending on the antigen co-administered, we evaluated antipeptide antibody responses in serum and mucosal samples (vaginal, intestinal, and pulmonary) of mice that were immunized by intranasal or intraperitoneal routes with one of two distinct hybrid C4/V3 HIV peptides, either alone or with CT or Cry1Ac. The tested HIV 1 peptides differed in two aminoacids, T1SP10MN(A) was modified at the SP10 region by the substitution of the isoleucines 12 and 14 for cysteines and was called modified (m)T1SP10MN(A). Our data indicate that the adjuvant effects of CT and Cry1Ac are different. In addition they vary depending on the antigen co-administered and the number of antigen doses, because after three doses moderate adjuvant effects of CT and Cry1Ac on anti-peptide serum and mucosal antibody responses were observed only against the mT1SP10MN(A). In contrast, to attain significant adjuvant effects against the T1SP10MN, four doses were required. Interestingly we found that modification of the HIV peptide increases its immunogenicity without altering the principal neutralizing determinant (SP10).

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

PubMed

Peters, Haley L; Tripathi, Satyendra C; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R; St John, Lisa S; Federico, Lorenzo; Meraz, Ismail M; Roth, Jack A; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L; Heymach, John V; Swisher, Stephen G; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J

2017-04-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR . ©2017 American Association for Cancer Research.

Selective induction of cell-mediated immunity and protection of rhesus macaques from chronic SHIV{sub KU2} infection by prophylactic vaccination with a conserved HIV-1 envelope peptide-cocktail

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

Nehete, Pramod N.; Nehete, Bharti P.; Hill, Lori

2008-01-05

Infection of Indian-origin rhesus macaques by the simian human immunodeficiency virus (SHIV) is considered to be a suitable preclinical model for directly testing efficacy of vaccine candidates based on the HIV-1 envelope. We used this model for prophylactic vaccination with a peptide-cocktail comprised of highly conserved HIV-1 envelope sequences immunogenic/antigenic in macaques and humans. Separate groups of macaques were immunized with the peptide-cocktail by intravenous and subcutaneous routes using autologous dendritic cells (DC) and Freund's adjuvant, respectively. The vaccine elicited antigen specific IFN-{gamma}-producing cells and T-cell proliferation, but not HIV-neutralizing antibodies. The vaccinated animals also exhibited efficient cross-clade cytolytic activitymore » against target cells expressing envelope proteins corresponding to HIV-1 strains representative of multiple clades that increased after intravenous challenge with pathogenic SHIV{sub KU2}. Virus-neutralizing antibodies were either undetectable or present only transiently at low levels in the control as well as vaccinated monkeys after infection. Significant control of plasma viremia leading to undetectable levels was achieved in majority of vaccinated monkeys compared to mock-vaccinated controls. Monkeys vaccinated with the peptide-cocktail using autologous DC, compared to Freund's adjuvant, and the mock-vaccinated animals, showed significantly higher IFN-{gamma} production, higher levels of vaccine-specific IFN-{gamma} producing CD4{sup +} cells and significant control of plasma viremia. These results support DC-based vaccine delivery and the utility of the conserved HIV-1 envelope peptide-cocktail, capable of priming strong cell-mediated immunity, for potential inclusion in HIV vaccination strategies.« less

Prediction and analysis of promiscuous T cell-epitopes derived from the vaccine candidate antigens of Leishmania donovani binding to MHC class-II alleles using in silico approach.

PubMed

Kashyap, Manju; Jaiswal, Varun; Farooq, Umar

2017-09-01

Visceral leishmaniasis is a dreadful infectious disease and caused by the intracellular protozoan parasites, Leishmania donovani and Leishmania infantum. Despite extensive efforts for developing effective prophylactic vaccine, still no vaccine is available against leishmaniasis. However, advancement in immunoinformatics methods generated new dimension in peptide based vaccine development. The present study was aimed to identify T-cell epitopes from the vaccine candidate antigens like Lipophosphogylcan-3(LPG-3) and Nucleoside hydrolase (NH) from the L. donovani using in silico methods. Available best tools were used for the identification of promiscuous peptides for MHC class-II alleles. A total of 34 promiscuous peptides from LPG-3, 3 from NH were identified on the basis of their 100% binding affinity towards all six HLA alleles, taken in this study. These peptides were further checked computationally to know their IFN-γ and IL4 inducing potential and nine peptides were identified. Peptide binding interactions with predominant HLA alleles were done by docking. Out of nine docked promiscuous peptides, only two peptides (QESRILRVIKKKLVR, RILRVIKKKLVRKTL), from LPG-3 and one peptide (FDKFWCLVIDALKRI) from NH showed lowest binding energy with all six alleles. These promiscuous T-cell epitopes were predicted on the basis of their antigenicity, hydrophobicity, potential immune response and docking scores. The immunogenicity of predicted promiscuous peptides might be used for subunit vaccine development with immune-modulating adjuvants. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-08-01

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

Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells.

PubMed

Corman, J M; Sercarz, E E; Nanda, N K

1998-11-01

It is now becoming accepted that one is not tolerant to all the determinants of self proteins: the T cell repertoire directed to some sequences in self proteins is intact and can be activated. When a self protein is exclusively expressed by tumour cells, the T cell repertoire directed to the particular self antigen can potentially be activated to attack the tumour: this would amount to induction of a beneficial autoimmune response. Prostate cancer offers a unique opportunity for activation of a tumour-specific immune response owing to the exclusive synthesis of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) by prostatic tissue and prostate tumour cells. In this study we examine the CD4 and CD8 T cell repertoires specific for peptides of PSA and PSM in normal human male individuals, using short-term, peptide antigen-driven CD4 and CD8 T cell lines. We show that short-term, CD4 T cell lines derived from six HLA-DR4 individuals showed strong proliferative responses to six of 10 tested peptides of PSA, selected as to contain a DR4 binding motif. Short-term, CD8 T cell lines from three HLA-A1 individuals showed specific cytolytic activity for autologous targets loaded with five of five tested peptides of PSA and PSM, selected to possess an HLA-A1 binding motif. One of the peptides chosen is termed a 'dual-motif' peptide, as it encodes determinants for both CD4 and CD8 T cells. These results, indicating the existence of CD4 and CD8 T cells against determinants of the self proteins, PSA and PSM, in healthy male individuals reveal the potential of the T cell repertoire from the typical prostate cancer patient to eradicate prostate tumours upon being appropriately activated.

Enhanced immune response to gastric cancer specific antigen Peptide by coencapsulation with CpG oligodeoxynucleotides in nanoemulsion.

PubMed

Shi, Rui; Hong, Liu; Wu, Daocheng; Ning, Xiaoxuan; Chen, Yu; Lin, Tao; Fan, Daiming; Wu, Kaichun

2005-02-01

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to construct a nanovaccine coencapsulated with a gastric cancer specific antigen MG7 mimotope peptide and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. It was shown that the tumor masses formed in the mice immunized with coencapsulated nanovaccine (0.0825 g) markedly smaller (P < 0.01) than those formed in the mice immunized with nanovaccine encapsulated with antigen peptide alone (0.4465 g). A tumor inhibiting rate as high as 82.5% of the coencapsulated nanovaccine was obtained, while nanovaccine encapsulated with peptide only could not achieve the same effect (28.5%) (P < 0.01). Enzyme-linked immunospot assay (ELISPOT) showed that immunization using MG7 mimotope peptide coencapsulated with CpG ODN within the same nanoemulsion enhanced the frequency of splenocytes secreting IFN-gamma significantly (P < 0.01) when compared with immunization using MG7 peptide encapsulated in nanoemulsion alone (197spots/1 x 10(6) vs. 73 spots/1 x 10(6)). Cellular ELISA indicated that serum titer of antibody against MG7-Ag was significantly higher (P < 0.01) in mice immunized with coencapsulation form nanovaccine (0.7884) than that in the group immunized with nanovaccine encapsulated with MG7 peptide alone (0.3616). Using intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD4+ T-cells. Our experiments suggest that a vaccinal approach using nano-delivery system carrying in tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.

Definition of neutralizing sites on African horse sickness virus serotype 4 VP2 at the level of peptides.

PubMed

Martínez-Torrecuadrada, J L; Langeveld, J P; Meloen, R H; Casal, J I

2001-10-01

The antigenic structure of African horse sickness virus (AHSV) serotype 4 capsid protein VP2 has been determined at the peptide level by PEPSCAN analysis in combination with a large collection of polyclonal antisera and monoclonal antibodies. VP2, the determinant for the virus serotype and an important target in virus neutralization, was found to contain 15 antigenic sites. A major antigenic region containing 12 of the 15 sites was identified in the region between residues 223 and 400. A second domain between residues 568 and 681 contained the three remaining sites. These sites were used for the synthesis of peptides, which were later tested in rabbits. Of the 15 synthetic peptides, three were able to induce neutralizing antibodies for AHSV-4, defining two neutralizing epitopes, 'a' and 'b', between residues 321 and 339, and 377 and 400, respectively. A combination of peptides representing both sites induced a more effective neutralizing response. Still, the relatively low neutralization titres make the possibility of producing a synthetic vaccine for AHSV unlikely. The complex protein-protein interaction of the outer shell of the viral capsid would probably require the presence of either synthetic peptides in the correct conformation or peptide segments from the different proteins VP2, VP5 and VP7.

Adaptor protein-3 is required in dendritic cells for optimal Toll-like receptor signaling from phagosomes and antigen presentation to CD4+ T cells

PubMed Central

Mantegazza, Adriana R.; Guttentag, Susan H.; El-Benna, Jamel; Sasai, Miwa; Iwasaki, Akiko; Shen, Hao; Laufer, Terri M.; Marks, Michael S.

2012-01-01

SUMMARY Effective major histocompatibility complex-II (MHC-II) antigen presentation from phagocytosed particles requires phagosome-intrinsic toll-like receptor (TLR) signaling, but the molecular mechanisms underlying TLR delivery to phagosomes and how signaling regulates antigen presentation are incompletely understood. We show a requirement in dendritic cells (DCs) for adaptor protein-3 (AP-3) in efficient TLR recruitment to phagosomes and MHC-II presentation of antigens internalized by phagocytosis but not receptor-mediated endocytosis. DCs from AP-3-deficient pearl mice elicited impaired CD4+ T cell activation and Th1 effector function to particulate antigen in vitro and to recombinant Listeria monocytogenes infection in vivo. Whereas phagolysosome maturation and peptide:MHC-II complex assembly proceeded normally in pearl DCs, peptide:MHC-II export to the cell surface was impeded. This correlated with reduced TLR4 recruitment and proinflammatory signaling from phagosomes by particulate TLR ligands. We propose that AP-3-dependent TLR delivery from endosomes to phagosomes and subsequent signaling mobilize peptide:MHC-II export from intracellular stores. PMID:22560444

AgdbNet – antigen sequence database software for bacterial typing

PubMed Central

Jolley, Keith A; Maiden, Martin CJ

2006-01-01

Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated. PMID:16790057

Abacavir induces loading of novel self-peptides into HLA-B*57:01: an autoimmune model for HLA-associated drug hypersensitivity

PubMed Central

Norcross, Michael A.; Luo, Shen; Lu, Li; Boyne, Michael T.; Gomarteli, Mary; Rennels, Aaron D.; Woodcock, Janet; Margulies, David H.; McMurtrey, Curtis; Vernon, Stephen; Hildebrand, William H.; Buchli, Rico

2014-01-01

Background Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B*57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B*57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B*57:01 from abacavir-treated cells. Design and methods An HLA-B*57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B*57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B*57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B*57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B*57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B*57:01 with high affinity that was not altered by abacavir addition. Conclusion Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B*57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity. PMID:22617051

Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity.

PubMed

Norcross, Michael A; Luo, Shen; Lu, Li; Boyne, Michael T; Gomarteli, Mary; Rennels, Aaron D; Woodcock, Janet; Margulies, David H; McMurtrey, Curtis; Vernon, Stephen; Hildebrand, William H; Buchli, Rico

2012-07-17

Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B57:01 from abacavir-treated cells. An HLA-B57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B57:01 with high affinity that was not altered by abacavir addition. Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

PubMed

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Mimicry of the immunodominant conformation-dependent antigenic site of hepatitis A virus by motifs selected from synthetic peptide libraries.

PubMed

Mattioli, S; Imberti, L; Stellini, R; Primi, D

1995-09-01

Hepatitis A virus (HAV) is a positive-strand RNA virus with a genome length of approximately 7,480 nucleotides. Although HAV morphogenesis is thought to be similar to that of poliovirus, the prototype picornavirus, the complete characterization of the antigenic structure of this virus remains elusive. All the available evidences, however, support the existence, on HAV virions and empty capsids, of an immunodominant neutralization antigenic site which is conformation dependent and whose structure involves residues of both VP1 and VP3 capsid proteins. This particular feature and the difficulty of obtaining high virus yield in tissue cultures make HAV an ideal target for developing synthetic peptides that simulate the structure of its main antigenic determinant. To this end we utilized, in the present work, the divide-couple-recombine approach to generate a random library composed of millions of different hexapeptides. This vast library was screened with a well-characterized anti-HAV monoclonal antibody. By this strategy we identified a peptide that reacted specifically with monoclonal and polyclonal anti-HAV antibodies and, in mice, induced a specific anti-virus immune response. Furthermore, the peptide could also be used in an enzyme-linked immunosorbent assay for revealing a primary immunoglobulin M immune response in sera of acutely infected human patients. Interestingly, no sequence homology was found between the identified peptide and the HAV capsid proteins VP1 and VP3. Collectively, these data represent an additional important paradigm of a mimotope capable of mimicking an antigenic determinant with unknown tertiary structure.

Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated β-Turn Structures to be Used as Probes in Autoimmune Diseases

NASA Astrophysics Data System (ADS)

Giangrande, Chiara; Auberger, Nicolas; Rentier, Cédric; Papini, Anna Maria; Mallet, Jean-Maurice; Lavielle, Solange; Vinh, Joëlle

2016-04-01

Synthetic sugar-modified peptides were identified as antigenic probes in the context of autoimmune diseases. The aim of this work is to provide a mechanistic study on the fragmentation of different glycosylated analogs of a synthetic antigenic probe able to detect antibodies in a subpopulation of multiple sclerosis patients. In particular the N-glucosylated type I' β-turn peptide structure called CSF114(Glc) was used as a model to find signature fragmentations exploring the potential of multi-stage mass spectrometry by MALDI-LTQ Orbitrap. Here we compare the fragmentation of the glucosylated form of the synthetic peptide CSF114(Glc), bearing a glucose moiety on an asparagine residue, with less or non- immunoreactive forms, bearing different sugar-modifications, such as CSF114(GlcNAc), modified with a residue of N-acetylglucosamine, and CSF114[Lys7(1-deoxyfructopyranosyl)], this last one modified with a 1-deoxyfructopyranosyl moiety on a lysine at position 7. The analysis was set up using a synthetic compound specifically deuterated on the C-1 to compare its fragmentation with the fragmentation of the undeuterated form, and thus ascertain with confidence the presence on an Asn(Glc) within a peptide sequence. At the end of the study, our analysis led to the identification of signature neutral losses inside the sugar moieties to characterize the different types of glycosylation/glycation. The interest of this study lies in the possibility of applyimg this approach to the discovery of biomarkers and in the diagnosis of autoimmune diseases.

Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated β-Turn Structures to be Used as Probes in Autoimmune Diseases.

PubMed

Giangrande, Chiara; Auberger, Nicolas; Rentier, Cédric; Papini, Anna Maria; Mallet, Jean-Maurice; Lavielle, Solange; Vinh, Joëlle

2016-04-01

Synthetic sugar-modified peptides were identified as antigenic probes in the context of autoimmune diseases. The aim of this work is to provide a mechanistic study on the fragmentation of different glycosylated analogs of a synthetic antigenic probe able to detect antibodies in a subpopulation of multiple sclerosis patients. In particular the N-glucosylated type I' β-turn peptide structure called CSF114(Glc) was used as a model to find signature fragmentations exploring the potential of multi-stage mass spectrometry by MALDI-LTQ Orbitrap. Here we compare the fragmentation of the glucosylated form of the synthetic peptide CSF114(Glc), bearing a glucose moiety on an asparagine residue, with less or non- immunoreactive forms, bearing different sugar-modifications, such as CSF114(GlcNAc), modified with a residue of N-acetylglucosamine, and CSF114[Lys(7)(1-deoxyfructopyranosyl)], this last one modified with a 1-deoxyfructopyranosyl moiety on a lysine at position 7. The analysis was set up using a synthetic compound specifically deuterated on the C-1 to compare its fragmentation with the fragmentation of the undeuterated form, and thus ascertain with confidence the presence on an Asn(Glc) within a peptide sequence. At the end of the study, our analysis led to the identification of signature neutral losses inside the sugar moieties to characterize the different types of glycosylation/glycation. The interest of this study lies in the possibility of applyimg this approach to the discovery of biomarkers and in the diagnosis of autoimmune diseases. Graphical Abstract .

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

PubMed Central

McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

2010-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4+ T cell responses in cattle. Monocytes and CD4+ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4+ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4+ T cell responses in vitro for an important pathogen of livestock. PMID:20167197

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells.

PubMed

Ito, Masaki; Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2017-01-01

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: "physical adjuvants" increase the efficacy of antigen presentation by antigen-presenting cells (APC) and "signal adjuvants" induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create "adjuvant-free" antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif's function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens.

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells

PubMed Central

Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2017-01-01

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: “physical adjuvants” increase the efficacy of antigen presentation by antigen-presenting cells (APC) and “signal adjuvants” induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create “adjuvant-free” antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif’s function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens. PMID:29190754

Deciphering complex patterns of class-I HLA-peptide cross-reactivity via hierarchical grouping.

PubMed

Mukherjee, Sumanta; Warwicker, Jim; Chandra, Nagasuma

2015-07-01

T-cell responses in humans are initiated by the binding of a peptide antigen to a human leukocyte antigen (HLA) molecule. The peptide-HLA complex then recruits an appropriate T cell, leading to cell-mediated immunity. More than 2000 HLA class-I alleles are known in humans, and they vary only in their peptide-binding grooves. The polymorphism they exhibit enables them to bind a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern, as many peptides bind to a given allele and a given peptide can be recognized by many alleles. A powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. We present a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification was obtained, with 7 supergroups, each further subclassifying to yield 72 groups. An independent clustering performed based only on similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explain the basis of clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues contributing to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants.

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

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

Luo, Heng; Ye, Hao; Ng, Hui Wen

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics.

PubMed

Gul, Ahmet; Erman, Burak

2018-01-16

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

PubMed Central

Luo, Heng; Ye, Hao; Ng, Hui Wen; Sakkiah, Sugunadevi; Mendrick, Donna L.; Hong, Huixiao

2016-01-01

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system. PMID:27558848

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

DOE PAGES

Luo, Heng; Ye, Hao; Ng, Hui Wen; ...

2016-08-25

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

NASA Astrophysics Data System (ADS)

Gul, Ahmet; Erman, Burak

2018-03-01

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

Different antigen processing activities in dendritic cells, macrophages and monocytes lead to uneven production of HIV epitopes and affect CTL recognition

PubMed Central

Duong, Ellen; Bracho-Sanchez, Edith; Rucevic, Marijana; Liebesny, Paul H.; Xu, Yang; Shimada, Mariko; Ghebremichael, Musie; Kavanagh, Daniel G.; Le Gall, Sylvie

2014-01-01

Dendritic cells (DCs), macrophages (MPs) and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous antigens preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum (ER) translocation, trimming and MHC-I presentation. Here we compared the capacity of DCs, MPs and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848 and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs and monocytes. Differences in antigen processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load. PMID:25230751

Polyvalent immunoglobulin binding is an obstacle to accurate measurement of specific antibodies with ELISA despite inclusion of blocking agents.

PubMed

Loeffler, David A; Klaver, Andrea C

2017-11-01

Specific antibody concentrations are frequently measured in serum (and plasma and intravenous immunoglobulin) samples by enzyme-linked immunosorbent assay (ELISA). The standard negative control involves incubation of buffer alone on antigen-coated wells. The immunoreactivity that develops in antigen-coated wells in which diluted serum has been incubated is assumed to represent specific antibody binding. This approach can result in marked overestimation of specific antibody levels, because serum contains specific polyvalent antibodies which bind, primarily with low affinity, to multiple antigens (including those on ELISA plates) despite the use of blocking agents. Non-denaturing purification of serum IgG, followed by assessment of the antigen binding or antigen-binding affinity of this purified IgG, can reduce but not eliminate the problem of polyvalent antibody binding in indirect ELISAs. Alternatively, polyvalent antibody binding can be estimated by incubating a diluted serum sample on wells coated with an irrelevant protein (such as bovine serum albumin or a scrambled peptide sequence) or buffer alone, then subtracting this reactivity from the sample's binding to wells coated with the antigen of interest. Polyvalent binding of immunoglobulins must be accounted for in order to obtain accurate ELISA measurements of serum, plasma, or intravenous immunoglobulin antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

Knowledge-based grouping of modeled HLA peptide complexes.

PubMed

Kangueane, P; Sakharkar, M K; Lim, K S; Hao, H; Lin, K; Chee, R E; Kolatkar, P R

2000-05-01

Human leukocyte antigens are the most polymorphic of human genes and multiple sequence alignment shows that such polymorphisms are clustered in the functional peptide binding domains. Because of such polymorphism among the peptide binding residues, the prediction of peptides that bind to specific HLA molecules is very difficult. In recent years two different types of computer based prediction methods have been developed and both the methods have their own advantages and disadvantages. The nonavailability of allele specific binding data restricts the use of knowledge-based prediction methods for a wide range of HLA alleles. Alternatively, the modeling scheme appears to be a promising predictive tool for the selection of peptides that bind to specific HLA molecules. The scoring of the modeled HLA-peptide complexes is a major concern. The use of knowledge based rules (van der Waals clashes and solvent exposed hydrophobic residues) to distinguish binders from nonbinders is applied in the present study. The rules based on (1) number of observed atomic clashes between the modeled peptide and the HLA structure, and (2) number of solvent exposed hydrophobic residues on the modeled peptide effectively discriminate experimentally known binders from poor/nonbinders. Solved crystal complexes show no vdW Clash (vdWC) in 95% cases and no solvent exposed hydrophobic peptide residues (SEHPR) were seen in 86% cases. In our attempt to compare experimental binding data with the predicted scores by this scoring scheme, 77% of the peptides are correctly grouped as good binders with a sensitivity of 71%.

Cancer Immunotherapy Utilized Bubble Liposomes and Ultrasound as Antigen Delivery System

NASA Astrophysics Data System (ADS)

Oda, Yusuke; Otake, Shota; Suzuki, Ryo; Otake, Shota; Nishiie, Norihito; Hirata, Keiichi; Taira, Yuichiro; Utoguchi, Naoki; Maruyama, Kazuo

2010-03-01

In dendritic cells (DCs)-based cancer immunotherapy, it is important to present the epitope peptide derived from tumor associated antigens (TAAs) on MHC class I in order to induce tumor specific cytotoxic T lymphocytes (CTLs). However, MHC class I molecules generally present the epitope peptides derived from endogenous antigens for DCs but not exogenous ones such as TAAs. Recently, we developed the novel liposomal bubbles (Bubble liposomes) encapsulating perfluoropropane nanobubbles. In this study, we attempted to establish the novel antigen delivery system to induce MHC class I presentation using the combination of ultrasound and Bubble liposomes. Using ovalbumin (OVA) as model antigen, the combination of Bubble liposomes and ultrasound exposure for the DC could induce MHC class I presentation. In addition, the viability of DCs was more than 80%. These results suggest that Bubble liposomes might be a novel ultrasound enhanced antigen delivery tool in DC-based cancer immunotherapy.

PLGA particulate delivery systems for subunit vaccines: Linking particle properties to immunogenicity.

PubMed

Silva, A L; Soema, P C; Slütter, B; Ossendorp, F; Jiskoot, W

2016-04-02

Among the emerging subunit vaccines are recombinant protein- and synthetic peptide-based vaccine formulations. However, proteins and peptides have a low intrinsic immunogenicity. A common strategy to overcome this is to co-deliver (an) antigen(s) with (an) immune modulator(s) by co-encapsulating them in a particulate delivery system, such as poly(lactic-co-glycolic acid) (PLGA) particles. Particulate PLGA formulations offer many advantages for antigen delivery as they are biocompatible and biodegradable; can protect the antigens from degradation and clearance; allow for co-encapsulation of antigens and immune modulators; can be targeted to antigen presenting cells; and their particulate nature can increase uptake and cross-presentation by mimicking the size and shape of an invading pathogen. In this review we discuss the pros and cons of using PLGA particulate formulations for subunit vaccine delivery and provide an overview of formulation parameters that influence their adjuvanticity and the ensuing immune response.

The tyrosine phosphatase PTPN22 discriminates weak self peptides from strong agonist TCR signals.

PubMed

Salmond, Robert J; Brownlie, Rebecca J; Morrison, Vicky L; Zamoyska, Rose

2014-09-01

T cells must be tolerant of self antigens to avoid autoimmunity but responsive to foreign antigens to provide protection against infection. We found that in both naive T cells and effector T cells, the tyrosine phosphatase PTPN22 limited signaling via the T cell antigen receptor (TCR) by weak agonists and self antigens while not impeding responses to strong agonist antigens. T cells lacking PTPN22 showed enhanced formation of conjugates with antigen-presenting cells pulsed with weak peptides, which led to activation of the T cells and their production of inflammatory cytokines. This effect was exacerbated under conditions of lymphopenia, with the formation of potent memory T cells in the absence of PTPN22. Our data address how loss-of-function PTPN22 alleles can lead to the population expansion of effector and/or memory T cells and a predisposition to human autoimmunity.

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.

A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3

PubMed Central

Coulie, Pierre G.; Karanikas, Vaios; Colau, Didier; Lurquin, Christophe; Landry, Claire; Marchand, Marie; Dorval, Thierry; Brichard, Vincent; Boon, Thierry

2001-01-01

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis after in vitro restimulation indicated that about 1/40,000 postimmunization CD8+ blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 × 106 CD8+ lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection. PMID:11517302

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

PubMed

Thomas, P; Petrick, A T; Toth, C A; Fox, E S; Elting, J J; Steele, G

1992-10-30

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.

A facile approach to enhance antigen response for personalized cancer vaccination

NASA Astrophysics Data System (ADS)

Li, Aileen Weiwei; Sobral, Miguel C.; Badrinath, Soumya; Choi, Youngjin; Graveline, Amanda; Stafford, Alexander G.; Weaver, James C.; Dellacherie, Maxence O.; Shih, Ting-Yu; Ali, Omar A.; Kim, Jaeyun; Wucherpfennig, Kai W.; Mooney, David J.

2018-06-01

Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.

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.

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

Gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides

NASA Astrophysics Data System (ADS)

Tao, Yu; Zhang, Yan; Ju, Enguo; Ren, Hui; Ren, Jinsong

2015-07-01

We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments.We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02240a

The Specificity of Trimming of MHC Class I-Presented Peptides in the Endoplasmic Reticulum1

PubMed Central

Hearn, Arron; York, Ian A.; Rock, Kenneth L.

2010-01-01

Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell free biochemical system and in intact cells, we elucidated the specificity of ERAP1 and of ER trimming overall. ERAP1 can cleave after many amino acids on the N-terminus of epitope precursors but does so at markedly different rates. The specificity seen with purified ERAP1 is similar to that observed for trimming and presentation of epitopes in the ER of intact cells. We define N-terminal sequences that are favorable or unfavorable for antigen presentation in ways that are independent from the epitopes core sequence. When databases of known presented peptides were analyzed, the residues that were preferred for the trimming of model peptide precursors were found to be overrepresented in N-terminal flanking sequences of epitopes generally. These data define key determinants in the specificity of antigen processing. PMID:19828632

Site-specific incorporation of three toll-like receptor 2 targeting adjuvants into semisynthetic, molecularly defined nanoparticles: application to group a streptococcal vaccines.

PubMed

Moyle, Peter M; Dai, Wei; Zhang, Yingkai; Batzloff, Michael R; Good, Michael F; Toth, Istvan

2014-05-21

Subunit vaccines offer a means to produce safer, more defined vaccines compared to traditional whole microorganism approaches. Subunit antigens, however, exhibit weak immunity, which is normally overcome through coadministration with adjuvants. Enhanced vaccine properties (e.g., improved potency) can be obtained by linking antigen and adjuvant, as observed for synthetic peptide antigens and Toll-like receptor 2 (TLR2) ligands. As few protective peptide antigens have been reported, compared to protein antigens, we sought to extend the utility of this approach to recombinant proteins, while ensuring that conjugation reactions yielded a single, molecularly defined product. Herein we describe the development and optimization of techniques that enable the efficient, site-specific attachment of three synthetic TLR2 ligands (lipid core peptide (LCP), Pam2Cys, and Pam3Cys) onto engineered protein antigens, permitting the selection of optimal TLR2 agonists during the vaccine development process. Using this approach, broadly protective (J14) and population targeted (seven M protein N-terminal antigens) multiantigenic vaccines against group A streptococcus (GAS; Streptococcus pyogenes) were produced and observed to self-assemble in PBS to yield nanoparticules (69, 101, and 123 nm, respectively). All nanoparticle formulations exhibited self-adjuvanting properties, with rapid, persistent, antigen-specific IgG antibody responses elicited toward each antigen in subcutaneously immunized C57BL/6J mice. These antibodies were demonstrated to strongly bind to the cell surface of five GAS serotypes that are not represented by vaccine M protein N-terminal antigens, are among the top 20 circulating strains in developed countries, and are associated with clinical disease, suggesting that these vaccines may elicit broadly protective immune responses.

Establishment of HLA-DR4 Transgenic Mice for the Identification of CD4+ T Cell Epitopes of Tumor-Associated Antigens

PubMed Central

Harada, Kumiko; Michibata, Yayoi; Tsukamoto, Hirotake; Senju, Satoru; Tomita, Yusuke; Yuno, Akira; Hirayama, Masatoshi; Abu Sayem, Mohammad; Takeda, Naoki; Shibuya, Isao; Sogo, Shinji; Fujiki, Fumihiro; Sugiyama, Haruo; Eto, Masatoshi; Nishimura, Yasuharu

2013-01-01

Reports have shown that activation of tumor-specific CD4+ helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-Ed, where I-Ed α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-Ed has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination. PMID:24386437

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

PubMed Central

Pujol-Autonell, Irma; Serracant-Prat, Arnau; Cano-Sarabia, Mary; Ampudia, Rosa M.; Rodriguez-Fernandez, Silvia; Sanchez, Alex; Izquierdo, Cristina; Stratmann, Thomas; Puig-Domingo, Manuel; Maspoch, Daniel; Verdaguer, Joan; Vives-Pi, Marta

2015-01-01

Introduction The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. Objective To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. Methods A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. Results We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. Conclusions We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases. PMID:26039878

Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy

PubMed Central

2011-01-01

Background The mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance. Methods Fourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBV-specific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2. Results Interferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar. Conclusions There was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects. PMID:21320337

[Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide].

PubMed

Peng, Jing; Wang, Qiong; Cheng, Xiaoling; Liu, Mengwen; Wang, Mei; Xin, Huawei

2018-04-25

We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Use of autoantigen-loaded phosphatidylserine-liposomes to arrest autoimmunity in type 1 diabetes.

PubMed

Pujol-Autonell, Irma; Serracant-Prat, Arnau; Cano-Sarabia, Mary; Ampudia, Rosa M; Rodriguez-Fernandez, Silvia; Sanchez, Alex; Izquierdo, Cristina; Stratmann, Thomas; Puig-Domingo, Manuel; Maspoch, Daniel; Verdaguer, Joan; Vives-Pi, Marta

2015-01-01

The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes. A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.

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

PubMed

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

2000-04-01

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

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

PubMed Central

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A.; David, Chella S.; Admon, Arie; López, Daniel

2015-01-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. PMID:25635267

HPVdb: a data mining system for knowledge discovery in human papillomavirus with applications in T cell immunology and vaccinology

PubMed Central

Zhang, Guang Lan; Riemer, Angelika B.; Keskin, Derin B.; Chitkushev, Lou; Reinherz, Ellis L.; Brusic, Vladimir

2014-01-01

High-risk human papillomaviruses (HPVs) are the causes of many cancers, including cervical, anal, vulvar, vaginal, penile and oropharyngeal. To facilitate diagnosis, prognosis and characterization of these cancers, it is necessary to make full use of the immunological data on HPV available through publications, technical reports and databases. These data vary in granularity, quality and complexity. The extraction of knowledge from the vast amount of immunological data using data mining techniques remains a challenging task. To support integration of data and knowledge in virology and vaccinology, we developed a framework called KB-builder to streamline the development and deployment of web-accessible immunological knowledge systems. The framework consists of seven major functional modules, each facilitating a specific aspect of the knowledgebase construction process. Using KB-builder, we constructed the Human Papillomavirus T cell Antigen Database (HPVdb). It contains 2781 curated antigen entries of antigenic proteins derived from 18 genotypes of high-risk HPV and 18 genotypes of low-risk HPV. The HPVdb also catalogs 191 verified T cell epitopes and 45 verified human leukocyte antigen (HLA) ligands. Primary amino acid sequences of HPV antigens were collected and annotated from the UniProtKB. T cell epitopes and HLA ligands were collected from data mining of scientific literature and databases. The data were subject to extensive quality control (redundancy elimination, error detection and vocabulary consolidation). A set of computational tools for an in-depth analysis, such as sequence comparison using BLAST search, multiple alignments of antigens, classification of HPV types based on cancer risk, T cell epitope/HLA ligand visualization, T cell epitope/HLA ligand conservation analysis and sequence variability analysis, has been integrated within the HPVdb. Predicted Class I and Class II HLA binding peptides for 15 common HLA alleles are included in this database as putative targets. HPVdb is a knowledge-based system that integrates curated data and information with tailored analysis tools to facilitate data mining for HPV vaccinology and immunology. To our best knowledge, HPVdb is a unique data source providing a comprehensive list of HPV antigens and peptides. Database URL: http://cvc.dfci.harvard.edu/hpv/ PMID:24705205

HPVdb: a data mining system for knowledge discovery in human papillomavirus with applications in T cell immunology and vaccinology.

PubMed

Zhang, Guang Lan; Riemer, Angelika B; Keskin, Derin B; Chitkushev, Lou; Reinherz, Ellis L; Brusic, Vladimir

2014-01-01

High-risk human papillomaviruses (HPVs) are the causes of many cancers, including cervical, anal, vulvar, vaginal, penile and oropharyngeal. To facilitate diagnosis, prognosis and characterization of these cancers, it is necessary to make full use of the immunological data on HPV available through publications, technical reports and databases. These data vary in granularity, quality and complexity. The extraction of knowledge from the vast amount of immunological data using data mining techniques remains a challenging task. To support integration of data and knowledge in virology and vaccinology, we developed a framework called KB-builder to streamline the development and deployment of web-accessible immunological knowledge systems. The framework consists of seven major functional modules, each facilitating a specific aspect of the knowledgebase construction process. Using KB-builder, we constructed the Human Papillomavirus T cell Antigen Database (HPVdb). It contains 2781 curated antigen entries of antigenic proteins derived from 18 genotypes of high-risk HPV and 18 genotypes of low-risk HPV. The HPVdb also catalogs 191 verified T cell epitopes and 45 verified human leukocyte antigen (HLA) ligands. Primary amino acid sequences of HPV antigens were collected and annotated from the UniProtKB. T cell epitopes and HLA ligands were collected from data mining of scientific literature and databases. The data were subject to extensive quality control (redundancy elimination, error detection and vocabulary consolidation). A set of computational tools for an in-depth analysis, such as sequence comparison using BLAST search, multiple alignments of antigens, classification of HPV types based on cancer risk, T cell epitope/HLA ligand visualization, T cell epitope/HLA ligand conservation analysis and sequence variability analysis, has been integrated within the HPVdb. Predicted Class I and Class II HLA binding peptides for 15 common HLA alleles are included in this database as putative targets. HPVdb is a knowledge-based system that integrates curated data and information with tailored analysis tools to facilitate data mining for HPV vaccinology and immunology. To our best knowledge, HPVdb is a unique data source providing a comprehensive list of HPV antigens and peptides. Database URL: http://cvc.dfci.harvard.edu/hpv/.

Revisiting the arthritogenic peptide theory: quantitative not qualitative changes in the peptide repertoire of HLA-B27 allotypes.

PubMed

Schittenhelm, Ralf B; Sian, Terry C C Lim Kam; Wilmann, Pascal G; Dudek, Nadine L; Purcell, Anthony W

2015-03-01

The association of HLA-B27 with spondyloarthropathy is one of the strongest documented for any autoimmune disease. A common hypothesis for this association is the arthritogenic peptide concept. This dictates that differences in the peptide binding preferences of disease-associated and non-disease-associated HLA-B27 allotypes underlie the presentation of bacterial and self-peptides, leading to cross-reactive T cell immunity and subsequent autoimmune attack of affected tissues. The aim of this study was to analyze and compare self-peptides from 8 HLA-B27 allotypes, to increase existing data sets of HLA-B27 ligands, to refine and compare their consensus-binding motifs, and to reveal similarities and differences in the peptide repertoire of the HLA-B27 subtypes. Qualitative differences in the peptides bound to the 8 most frequent HLA-B27 subtypes were determined by tandem mass spectrometry, and quantitative changes in allelic binding specificities were determined by highly sensitive and targeted multiple reaction monitoring mass spectrometry. We identified >7,500 major histocompatibility complex class I peptides derived from the 8 most common HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09). We describe individual binding motifs for these alleles for the 9-12-mer ligands. The peptide repertoires of these closely related alleles showed significant overlap. Allelic polymorphisms resulting in changes in the amino acid composition of the antigen-binding cleft manifested largely as quantitative changes in the peptide cargo of these molecules. Absolute binding preferences of HLA-B27 allotypes do not explain disease association. The arthritogenic peptide theory needs to be reassessed in terms of quantitative changes in self-peptide presentation, T cell selection, and altered conformation of bound peptides. Copyright © 2015 by the American College of Rheumatology.

MHC structure and function − antigen presentation. Part 2

PubMed Central

Goldberg, Anna Carla; Rizzo, Luiz Vicente

2015-01-01

The second part of this review deals with the molecules and processes involved in the processing and presentation of the antigenic fragments to the T-cell receptor. Though the nature of the antigens presented varies, the most significant class of antigens is proteins, processed within the cell to be then recognized in the form of peptides, a mechanism that confers an extraordinary degree of precision to this mode of immune response. The efficiency and accuracy of this system is also the result of the myriad of mechanisms involved in the processing of proteins and production of peptides, in addition to the capture and recycling of alternative sources aiming to generate further diversity in the presentation to T-cells. PMID:25807243

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.

Immunization with 60 kD Ro peptide produces different stages of preclinical autoimmunity in a Sjögren's syndrome model among multiple strains of inbred mice.

PubMed

Kurien, B T; Dsouza, A; Igoe, A; Lee, Y J; Maier-Moore, J S; Gordon, T; Jackson, M; Scofield, R H

2013-07-01

Sjögren's syndrome is a chronic illness manifested characteristically by immune injury to the salivary and lacrimal glands, resulting in dry mouth/eyes. Anti-Ro [Sjögren's syndrome antigen A (SSA)] and anti-La [Sjögren's syndrome antigen B (SSB)] autoantibodies are found frequently in Sjögren's subjects as well as in individuals who will go on to develop the disease. Immunization of BALB/c mice with Ro60 peptides results in epitope spreading with anti-Ro and anti-La along with lymphocyte infiltration of salivary glands similar to human Sjögren's. In addition, these animals have poor salivary function/low saliva volume. In this study, we examined whether Ro-peptide immunization produces a Sjögren's-like illness in other strains of mice. BALB/c, DBA-2, PL/J, SJL/J and C57BL/6 mice were immunized with Ro60 peptide-274. Sera from these mice were studied by immunoblot and enzyme-linked immunosorbent assay for autoantibodies. Timed salivary flow was determined after pharmacological stimulation, and salivary glands were examined pathologically. We found that SJL/J mice had no immune response to the peptide from Ro60, while C57BL/6 mice produced antibodies that bound the peptide but had no epitope spreading. PL/J mice had epitope spreading to other structures of Ro60 as well as to La, but like C57BL/6 and SJL/J had no salivary gland lymphocytic infiltration and no decrement of salivary function. DBA-2 and BALB/c mice had infiltration but only BALB/c had decreased salivary function. The immunological processes leading to a Sjögren's-like illness after Ro-peptide immunization were interrupted in a stepwise fashion in these differing mice strains. These data suggest that this is a model of preclinical disease with genetic control for epitope spreading, lymphocytic infiltration and glandular dysfunction. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

A molecular view of multiple sclerosis and experimental autoimmune encephalitis: What can we learn from the epitope data?

PubMed Central

Vaughan, Kerrie; Peters, Bjoern; O'Connor, Kevin C.; Martin, Roland; Sette, Alessandro

2016-01-01

An analysis to inventory all immune epitope data related to multiple sclerosis (MS) was performed using the Immune Epitope Database (IEDB). The analysis revealed that MS related data represent >20% of all autoimmune data, and that studies of EAE predominate; only 22% of the references describe human data. To date, >5800 unique peptides, analogs, mimotopes, and/or non-protein epitopes have been reported from 861 references, including data describing myelin-containing, as well as non-myelin antigens. This work provides a reference point for the scientific community of the universe of available data for MS-related adaptive immunity in the context of EAE and human disease. PMID:24365494

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

PubMed Central

Bouvier, M; Wiley, D C

1996-01-01

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides. Images Fig. 2 Fig. 4 PMID:8643447

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

PubMed Central

Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects

PubMed Central

Tognon, Mauro; Corallini, Alfredo; Manfrini, Marco; Taronna, Angelo; Butel, Janet S.; Pietrobon, Silvia; Trevisiol, Lorenzo; Bononi, Ilaria; Vaccher, Emanuela; Barbanti-Brodano, Giuseppe; Martini, Fernanda; Mazzoni, Elisa

2016-01-01

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18–65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses. PMID:26731525

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

PubMed

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

2011-06-01

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

Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data*

PubMed Central

Schittenhelm, Ralf B.; Sivaneswaran, Saranjah; Lim Kam Sian, Terry C. C.; Croft, Nathan P.; Purcell, Anthony W.

2016-01-01

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s). PMID:26929215

The Kell protein of the common K2 phenotype is a catalytically active metalloprotease, whereas the rare Kell K1 antigen is inactive. Identification of novel substrates for the Kell protein.

PubMed

Clapéron, Audrey; Rose, Christiane; Gane, Pierre; Collec, Emmanuel; Bertrand, Olivier; Ouimet, Tanja

2005-06-03

The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.

Fine Specificity Mapping of Autoantigens Targeted by Anti-Centromere Autoantibodies

PubMed Central

Akbarali, Yasmin; Matousek-Ronck, Jennifer; Hunt, Laura; Staudt, Leslie; Reichlin, Morris; Guthridge, Joel M.; James, Judith A

2007-01-01

Summary Autoantibodies to centromeric proteins are commonly found in sera of limited scleroderma and other rheumatic disease patients. To better understand the inciting events and possible pathogenic mechanisms of these autoimmune responses, this study identified the common antigenic targets of CENP-A in scleroderma patient sera. Utilizing samples from 263 anti-centromere immunofluorescence positive patients, 93.5% were found to have anti-CENP-A reactivity and 95.4% had anti-CENP-B reactivity by ELISA. Very few patient samples exclusively targeted CENP-A (2.7%) or CENP-B (4.2%). Select patient sera were tested for reactivity with solid phase overlapping decapeptides of CENP-A. Four distinct epitopes of CENP-A were identified. Epitopes 2 and 3 were confirmed by additional testing of 263 patient sera by ELISA for reactivity with these sequences constructed as multiple antigenic peptides. Inhibition CENP-A Western blots also confirmed the specificity of these humoral peptide immune responses in a subset of patient sera. The first three arginine residues (aa 4-6) of CENP-A appear essential for antibody recognition, as replacing these arginines with glycine residues reduced antibody binding to the expressed CENP-A protein by an average of 93.2% (range 80-100%). In selected patients with serial samples spanning nearly a decade, humoral epitope binding patterns were quite stable and showed no epitope spreading over time. This epitope mapping study identifies key antigenic targets of the anti-centromere response and establishes that the majority of the responses depend on key amino-terminal residues. PMID:17210244

Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease

PubMed Central

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

2014-01-01

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

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

Human platelet antigen (HPA)-1a peptides do not reliably suppress anti-HPA-1a responses using a humanized severe combined immunodeficiency (SCID) mouse model

PubMed Central

Jackson, D J; Eastlake, J L; Kumpel, B M

2014-01-01

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs most frequently when human platelet antigen (HPA)-1a-positive fetal platelets are destroyed by maternal HPA-1a immunoglobulin (Ig)G antibodies. Pregnancies at risk are treated by administration of high-dose intravenous Ig (IVIG) to women, but this is expensive and often not well tolerated. Peptide immunotherapy may be effective for ameliorating some allergic and autoimmune diseases. The HPA-1a/1b polymorphism is Leu/Pro33 on β3 integrin (CD61), and the anti-HPA-1a response is restricted to HPA-1b1b and HLA-DRB3*0101-positive pregnant women with an HPA-1a-positive fetus. We investigated whether or not HPA-1a antigen-specific peptides that formed the T cell epitope could reduce IgG anti-HPA-1a responses, using a mouse model we had developed previously. Peripheral blood mononuclear cells (PBMC) in blood donations from HPA-1a-immunized women were injected intraperitoneally (i.p.) into severe combined immunodeficient (SCID) mice with peptides and HPA-1a-positive platelets. Human anti-HPA-1a in murine plasma was quantitated at intervals up to 15 weeks. HPA-1a-specific T cells in PBMC were identified by proliferation assays. Using PBMC of three donors who had little T cell reactivity to HPA-1a peptides in vitro, stimulation of anti-HPA-1a responses by these peptides occurred in vivo. However, with a second donation from one of these women which, uniquely, had high HPA-1a-specific T cell proliferation in vitro, marked suppression of the anti-HPA-1a response by HPA-1a peptides occurred in vivo. HPA-1a peptide immunotherapy in this model depended upon reactivation of HPA-1a T cell responses in the donor. For FNAIT, we suggest that administration of antigen-specific peptides to pregnant women might cause either enhancement or reduction of pathogenic antibodies. PMID:24261689

Ten tandem repeats of {beta}-hCG 109-118 enhance immunogenicity and anti-tumor effects of {beta}-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

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

Zhang Yankai; Yan Rong; He Yi

2006-07-14

The {beta}-subunit of human chorionic gonadotropin ({beta}-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of {beta}-hCG (109-118), then 10 tandemly repeated copies of the 10-residue sequence combined with {beta}-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-{beta}hCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residuemore » sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-{beta}hCGCTP37 and HSP65-{beta}hCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-{beta}hCGCTP37 elicited much higher levels of specific anti-{beta}-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-{beta}hCGCTP37, which should suggest that HSP65-X10-{beta}hCGCTP37 may be an effective protein vaccine for the treatment of {beta}-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.« less

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

PubMed

Gupta, Bhawna; Iancu, Emanuela M; Gannon, Philippe O; Wieckowski, Sébastien; Baitsch, Lukas; Speiser, Daniel E; Rufer, Nathalie

2012-07-01

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Design of ligand-targeted nanoparticles for enhanced cancer targeting

NASA Astrophysics Data System (ADS)

Stefanick, Jared F.

Ligand-targeted nanoparticles are increasingly used as drug delivery vehicles for cancer therapy, yet have not consistently produced successful clinical outcomes. Although these inconsistencies may arise from differences in disease models and target receptors, nanoparticle design parameters can significantly influence therapeutic efficacy. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, this work evaluates the roles of polyethylene glycol (PEG) coating, ethylene glycol (EG) peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size on tumor targeting in a systematic manner. These parameters were analyzed in multiple disease models by targeting human epidermal growth factor receptor 2 (HER2) in breast cancer and very late antigen-4 (VLA-4) in multiple myeloma to demonstrate the widespread applicability of this approach. By increasing the hydrophilicity of the targeting peptide sequence and simultaneously optimizing the EG peptide-linker length, the in vitro cellular uptake of targeted liposomes was significantly enhanced. Specifically, including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake ~80-fold using an EG6 peptide-linker compared to ~10-fold using an EG45 linker. In vivo, targeted liposomes prepared in a traditional manner lacking the oligolysine chain demonstrated similar biodistribution and tumor uptake to non-targeted liposomes. However, by including the oligolysine chain, targeted liposomes using an EG45 linker significantly improved tumor uptake ~8-fold over non-targeted liposomes, while the use of an EG6 linker decreased tumor accumulation and uptake, owing to differences in cellular uptake kinetics, clearance mechanisms, and binding site barrier effects. To further improve tumor targeting and enhance the selectivity of targeted nanoparticles, a dual-receptor targeted approach was evaluated by targeting multiple cell surface receptors simultaneously. Liposomes functionalized with two distinct peptide antagonists to target VLA-4 and Leukocyte Peyer's Patch Adhesion Molecule-1 (LPAM-1) demonstrated synergistically enhanced cellular uptake by cells overexpressing both target receptors and negligible uptake by cells that do not simultaneously express both receptors, providing a strategy to improve selectivity over conventional single receptor-targeted designs. Taken together, this process of systematic optimization of well-defined nanoparticle drug delivery systems has the potential to improve cancer therapy for a broader patient population.

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

PubMed

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A; David, Chella S; Admon, Arie; López, Daniel

2015-04-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mycobacterium tuberculosis region of difference (RD) 2 antigen Rv1985c and RD11 antigen Rv3425 have the promising potential to distinguish patients with active tuberculosis from M. bovis BCG-vaccinated individuals.

PubMed

Wang, Sen; Chen, Jiazhen; Zhang, Ying; Diao, Ni; Zhang, Shu; Wu, Jing; Lu, Chanyi; Wang, Feifei; Gao, Yan; Shao, Lingyun; Jin, Jialin; Weng, Xinhua; Zhang, Wenhong

2013-01-01

Antigens encoded in the region of difference (RD) of Mycobacterium tuberculosis constitute a potential source of specific immunodiagnostic antigens for distinguishing tuberculosis (TB) infection from BCG vaccination. We evaluated the diagnostic potential of specific T-cell epitopes selected from two immunodominant antigens, Rv1985c and Rv3425, from RD2 and RD11, respectively, on the basis of epitope mapping, in TB patients and BCG-vaccinated healthy individuals. Using a whole-blood gamma interferon release assay, a wide array of epitopes was recognized on both Rv1985c and Rv3425 in TB patients. Those epitopes that could specifically discriminate TB infection from BCG vaccination were carefully selected, and the most promising peptide pools from Rv1985c showed a sensitivity of 53.9% and a specificity of 95.5%. When the novel specific peptides from Rv1985c joined the diagnostic antigens in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, the sensitivity was increased from 86.4% to 96.2%, with no drop in specificity. These results indicate that the peptide pools selected from Rv1985c and Rv3425 have the potential to diagnose TB infection by a method that may be routinely used in clinical laboratories.

Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.

PubMed

Wang, Xiaona; Wang, Li; Huang, Xuewei; Ma, Sunting; Yu, Meiling; Shi, Wen; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

2017-10-25

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences ( p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

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

PubMed Central

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

2014-01-01

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

In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

PubMed Central

Tridente, Giuseppe; Bason, Caterina; Sivori, Simona; Beri, Ruggero; Dolcino, Marzia; Valletta, Enrico; Corrocher, Roberto; Puccetti, Antonio

2006-01-01

Background Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. Methods and Findings In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. Conclusions Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4. PMID:16984219

Immune privilege of the CNS is not the consequence of limited antigen sampling

NASA Astrophysics Data System (ADS)

Harris, Melissa G.; Hulseberg, Paul; Ling, Changying; Karman, Jozsef; Clarkson, Benjamin D.; Harding, Jeffrey S.; Zhang, Mengxue; Sandor, Adam; Christensen, Kelsey; Nagy, Andras; Sandor, Matyas; Fabry, Zsuzsanna

2014-03-01

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

PubMed

Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

2017-09-02

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

A lipid-based nano-regulator for cancer immunotherapy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Qian, Yuan; Qiao, Sha; Zhang, Zhihong

2017-02-01

In the application of nanotechnology in cancer immunotherapy, antigen presenting cells (APCs, dendritic cells and macrophages) are preferable target due to their endocytic capacity and suppressed phenotype. Recently, we developed a lipid-based core-shell nanocarrier, which is stabilized by changeable fusion peptides and possesses a sub-30 diameter. With the different peptides, the nanoparticles (NPs) could either target to dendritic cells (DCs) in lymph nodes (LNs) or tumor associated macrophages (TAMs) in tumor environment. After subcutaneous injection, the NPs could targeted deliver the encapsulated antigen peptides (APs) and adjuvants (CpG-ODN) to dendritic cells in LNs, and lead to the antigen presenting and activation of cytotoxic T lymphocytes against tumor. In other case, after systemic administration, the immune regulatory molecules were carried by NPs and targeting delivered to specific immunocytes in tumor microenvironment resulting in the immunosuppressive state broken and tumor growth inhibition.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

Administration of sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate conjugated GP100{sub 25–33} peptide-coupled spleen cells effectively mounts antigen-specific immune response against mouse melanoma

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

Chang, Xiaoli; Xia, Chang-Qing, E-mail: cqx65@yahoo.com; Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL32610

It remains a top research priority to develop immunotherapeutic approaches to induce potent antigen-specific immune responses against tumors. However, in spite of some promising results, most strategies are ineffective because they generate low numbers of tumor-reactive cytotoxic T lymphocytes (CTLs). Here we designed a strategy to enhance antigen-specific immune response via administering sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC)-conjugated melanoma tumor antigen GP100{sub 25–33} peptide-coupled syngeneic spleen cells in a mouse model of melanoma. We found that infusion of GP100{sub 25–33} peptide-coupled spleen cells significantly attenuated the growth of melanoma in prophylactic and therapeutic immunizations. Consistent with these findings, the adoptive transfer of spleenmore » cells from immunized mice to naïve syngeneic mice was able to transfer anti-tumor effect, suggesting that GP100{sub 25–33} peptide-specific immune response was induced. Further studies showed that, CD8+ T cell proliferation and the frequency of interferon (IFN)-γ-producing CD8+ T cells upon ex vivo stimulation by GP100{sub 25–33} were significantly increased compared to control groups. Tumor antigen, GP100{sub 25–23} specific immune response was also confirmed by ELISpot and GP100-tetramer assays. This approach is simple, easy-handled, and efficiently delivering antigens to lymphoid tissues. Our study offers an opportunity for clinically translating this approach into tumor immunotherapy. - Highlights: • Infusion of GP100{sub 25–33}-coupled spleen cells leads to potent anti-melanoma immunity. • GP100{sub 25–33}-coupled spleen cell treatment induces antigen-specific IFN-γ-producing CD8 T cells. • This approach takes advantage of homing nature of immune cells.« less

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

PubMed

Hiemstra, H S; van Veelen, P A; Schloot, N C; Geluk, A; van Meijgaarden, K E; Willemen, S J; Leunissen, J A; Benckhuijsen, W E; Amons, R; de Vries, R R; Roep, B O; Ottenhoff, T H; Drijfhout, J W

1998-10-15

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

In silico design of smart binders to anthrax PA

NASA Astrophysics Data System (ADS)

Sellers, Michael; Hurley, Margaret M.

2012-06-01

The development of smart peptide binders requires an understanding of the fundamental mechanisms of recognition which has remained an elusive grail of the research community for decades. Recent advances in automated discovery and synthetic library science provide a wealth of information to probe fundamental details of binding and facilitate the development of improved models for a priori prediction of affinity and specificity. Here we present the modeling portion of an iterative experimental/computational study to produce high affinity peptide binders to the Protective Antigen (PA) of Bacillus anthracis. The result is a general usage, HPC-oriented, python-based toolkit based upon powerful third-party freeware, which is designed to provide a better understanding of peptide-protein interactions and ultimately predict and measure new smart peptide binder candidates. We present an improved simulation protocol with flexible peptide docking to the Anthrax Protective Antigen, reported within the context of experimental data presented in a companion work.

Crystal Structures of HLA-A*0201 Complexed with Melan-A/MART-1[subscript 26(27L)-35] Peptidomimetics Reveal Conformational Heterogeneity and Highlight Degeneracy of T Cell Recognition

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

Douat-Casassus, Celine; Borbulevych, Oleg; Tarbe, Marion

2010-10-07

There is growing interest in using tumor associated antigens presented by class I major histocompatibility complex (MHC-I) proteins as cancer vaccines. As native peptides are poorly stable in biological fluids, researchers have sought to engineer synthetic peptidomimetics with greater biostability. Here, we demonstrate that antigenic peptidomimetics of the Melan-A/MART-1{sub 26(27L)-35} melanoma antigen adopt strikingly different conformations when bound to MHC-I, highlighting the degeneracy of T cell recognition and revealing the challenges associated with mimicking native peptide conformation.

A Novel Heat Shock Protein 70-based Vaccine Prepared from DC-Tumor Fusion Cells.

PubMed

Weng, Desheng; Calderwood, Stuart K; Gong, Jianlin

2018-01-01

We have developed an enhanced molecular chaperone-based vaccine through rapid isolation of Hsp70 peptide complexes after the fusion of tumor and dendritic cells (Hsp70.PC-F). In this approach, the tumor antigens are introduced into the antigen processing machinery of dendritic cells through the cell fusion process and thus we can obtain antigenic tumor peptides or their intermediates that have been processed by dendritic cells. Our results show that Hsp70.PC-F has increased immunogenicity compared to preparations from tumor cells alone and therefore constitutes an improved formulation of chaperone protein-based tumor vaccine.

The biology and underlying mechanisms of cross-presentation of exogenous antigens on MHC I molecules

PubMed Central

Cruz, Freidrich M.; Colbert, Jeff D.; Merino, Elena; Kriegsman, Barry A.; Rock, Kenneth L.

2017-01-01

To monitor the health of cells, the immune system tasks antigen presenting cells with gathering antigens from other cells and reporting them to CD8 T cells in the form of peptides bound to MHC I molecules. Most cells would be unable to perform this function because they use their MHC I molecules to exclusively present peptides derived from the cell’s own proteins. However, the immune system evolved mechanisms for dendritic cells and some other phagocytes to sample and present antigens from the extracellular milieu on MHC I through a process called cross-presentation (XPT). How this important task is accomplished, its role in health and disease and its potential for exploitation are the subject of this review. PMID:28125356

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

PubMed Central

BANNAI, Hiroshi; NEMOTO, Manabu; TSUJIMURA, Koji; YAMANAKA, Takashi; MAEDA, Ken; KONDO, Takashi

2015-01-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA. PMID:26424485

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

PubMed

Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

2016-02-01

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

PubMed

Jadhao, Samadhan J; Anderson, Larry J

2016-01-01

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

Polyclonal cell activity of a repeat peptide derived from the sequence of an 85-kilodalton surface protein of Trypanosoma cruzi trypomastigotes.

PubMed Central

Pestel, J; Defoort, J P; Gras-Masse, H; Afchain, D; Capron, A; Tartar, A; Ouaissi, A

1992-01-01

Some in vitro and in vivo biological activities of an octadecapeptide derived from an 85-kDa surface protein of Trypanosoma cruzi trypomastigote were studied. The peptide coupled to a carrier protein induced the proliferative response of lymph node cells from mice immunized with various antigens. Moreover, sera from mice immunized with the coupled peptide were found to contain antibodies against a number of self and nonself antigens: fibronectin, bovine serum albumin, myosin, tetanus toxoid, ovalbumin, keyhole limpet hemocyanin, and DNA. These results are discussed in the context of Chagas' disease immunopathology. PMID:1730508

T-cell memory responses elicited by yellow fever vaccine are targeted to overlapping epitopes containing multiple HLA-I and -II binding motifs.

PubMed

de Melo, Andréa Barbosa; Nascimento, Eduardo J M; Braga-Neto, Ulisses; Dhalia, Rafael; Silva, Ana Maria; Oelke, Mathias; Schneck, Jonathan P; Sidney, John; Sette, Alessandro; Montenegro, Silvia M L; Marques, Ernesto T A

2013-01-01

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4(+) and CD8(+) T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

PubMed Central

de Melo, Andréa Barbosa; Nascimento, Eduardo J. M.; Braga-Neto, Ulisses; Dhalia, Rafael; Silva, Ana Maria; Oelke, Mathias; Schneck, Jonathan P.; Sidney, John; Sette, Alessandro; Montenegro, Silvia M. L.; Marques, Ernesto T. A.

2013-01-01

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, “promiscuous” T-cell antigens might contribute to the high efficacy of the yellow fever vaccines. PMID:23383350

Viral Inhibition of the Transporter Associated with Antigen Processing (TAP): A Striking Example of Functional Convergent Evolution

PubMed Central

Verweij, Marieke C.; Horst, Daniëlle; Griffin, Bryan D.; Luteijn, Rutger D.; Davison, Andrew J.; Ressing, Maaike E.; Wiertz, Emmanuel J. H. J.

2015-01-01

Herpesviruses are large DNA viruses that are highly abundant within their host populations. Even in the presence of a healthy immune system, these viruses manage to cause lifelong infections. This persistence is partially mediated by the virus entering latency, a phase of infection characterized by limited viral protein expression. Moreover, herpesviruses have devoted a significant part of their coding capacity to immune evasion strategies. It is believed that the close coexistence of herpesviruses and their hosts has resulted in the evolution of viral proteins that specifically attack multiple arms of the host immune system. Cytotoxic T lymphocytes (CTLs) play an important role in antiviral immunity. CTLs recognize their target through viral peptides presented in the context of MHC molecules at the cell surface. Every herpesvirus studied to date encodes multiple immune evasion molecules that effectively interfere with specific steps of the MHC class I antigen presentation pathway. The transporter associated with antigen processing (TAP) plays a key role in the loading of viral peptides onto MHC class I molecules. This is reflected by the numerous ways herpesviruses have developed to block TAP function. In this review, we describe the characteristics and mechanisms of action of all known virus-encoded TAP inhibitors. Orthologs of these proteins encoded by related viruses are identified, and the conservation of TAP inhibition is discussed. A phylogenetic analysis of members of the family Herpesviridae is included to study the origin of these molecules. In addition, we discuss the characteristics of the first TAP inhibitor identified outside the herpesvirus family, namely, in cowpox virus. The strategies of TAP inhibition employed by viruses are very distinct and are likely to have been acquired independently during evolution. These findings and the recent discovery of a non-herpesvirus TAP inhibitor represent a striking example of functional convergent evolution. PMID:25880312

Quantitative impact of thymic selection on Foxp3+ and Foxp3- subsets of self-peptide/MHC class II-specific CD4+ T cells.

PubMed

Moon, James J; Dash, Pradyot; Oguin, Thomas H; McClaren, Jennifer L; Chu, H Hamlet; Thomas, Paul G; Jenkins, Marc K

2011-08-30

It is currently thought that T cells with specificity for self-peptide/MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4(+) T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3(+) regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4(+) T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3(-) cells, but also contained a subset of Foxp3(+) regulatory cells. Both Foxp3(-) and Foxp3(+) pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3(+) subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII-specific CD4(+) T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.

Bacteriophage-fused peptides for serodiagnosis of human strongyloidiasis.

PubMed

Feliciano, Nágilla Daliane; Ribeiro, Vanessa da Silva; Santos, Fabiana de Almeida Araújo; Fujimura, Patricia Tiemi; Gonzaga, Henrique Tomaz; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

2014-01-01

Strongyloidiasis, a human intestinal infection caused by the nematode Strongyloides stercoralis, is frequently underdiagnosed and although its high prevalence is still a neglected parasitic disease because conventional diagnostic tests based on parasitological examination (presence of Strongyloides larvae in stool) are not sufficiently sensitive due to the low parasitic load and to the irregular larval output. There is an urgent need to improve diagnostic assays, especially for immunocompromised patients with high parasitic load as consequence of self-infection cycle, which can disseminate throughout the body, resulting in a potentially fatal hyperinfection syndrome often accompanied by sepsis or meningitis. We have performed Phage Display technology to select peptides that mimic S. stercoralis antigens, capable of detecting a humoral response in patients with strongyloidiasis. The peptides reactivity was investigated by Phage-ELISA through different panels of serum samples. We have successfully selected five peptides with significant immunoreactivity to circulating IgG from patients' sera with strongyloidiasis. The phage displayed peptides C9 and C10 presented the highest diagnostic potential (AUC>0.87) with excellent sensitivity (>85%) and good specificity (>77.5%), suggesting that some S. stercoralis antigens trigger systemic immune response. These novel antigens are interesting serum biomarkers for routine strongyloidiasis screenings due to the easy production and simple assay using Phage-ELISA. Such markers may also present a promising application for therapeutic monitoring.

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs.

PubMed

Santos, Marcus Rebouças; Assao, Viviane Sisdelli; Santos, Fabiana de Almeida Araújo; Salgado, Rafael Locatelli; Carneiro, Ana Paula; Fietto, Juliana Lopes Rangel; Bressan, Gustavo Costa; de Almeida, Márcia Rogéria; Lobato, Zelia Inês Portela; Ueira-Veira, Carlos; Goulart, Luíz Ricardo; Silva-Júnior, Abelardo

2018-07-01

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

PubMed Central

Thibaut, Julien; Mérieux, Yves; Rigal, Dominique; Gillet, Germain

2012-01-01

Background Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays. Design and Methods Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a. Results This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies. Conclusions This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes. PMID:22133781

Systemic Lupus Erythematosus: Molecular Mimicry between Anti-dsDNA CDR3 Idiotype, Microbial and Self Peptides-As Antigens for Th Cells.

PubMed

Aas-Hanssen, Kristin; Thompson, Keith M; Bogen, Bjarne; Munthe, Ludvig A

2015-01-01

Systemic lupus erythematosus (SLE) is marked by a T helper (Th) cell-dependent B cell hyperresponsiveness, with frequent germinal center reactions, and gammaglobulinemia. A feature of SLE is the finding of IgG autoantibodies specific for dsDNA. The specificity of the Th cells that drive the expansion of anti-dsDNA B cells is unresolved. However, anti-microbial, anti-histone, and anti-idiotype Th cell responses have been hypothesized to play a role. It has been entirely unclear if these seemingly disparate Th cell responses and hypotheses could be related or unified. Here, we describe that H chain CDR3 idiotypes from IgG(+) B cells of lupus mice have sequence similarities with both microbial and self peptides. Matched sequences were more frequent within the mutated CDR3 repertoire and when sequences were derived from lupus mice with expanded anti-dsDNA B cells. Analyses of histone sequences showed that particular histone peptides were similar to VDJ junctions. Moreover, lupus mice had Th cell responses toward histone peptides similar to anti-dsDNA CDR3 sequences. The results suggest that Th cells in lupus may have multiple cross-reactive specificities linked to the IgVH CDR3 Id-peptide sequences as well as similar DNA-associated protein motifs.

Structural Elements Recognized by Abacavir-Induced T Cells.

PubMed

Yerly, Daniel; Pompeu, Yuri Andreiw; Schutte, Ryan J; Eriksson, Klara K; Strhyn, Anette; Bracey, Austin W; Buus, Soren; Ostrov, David A

2017-07-07

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976-984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230-238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues.

[Diagnostic values of type III Procollagen N-terminal peptide and combination assay of type III procollagen N-terminal peptide with CEA and CA 19-9 in gastric cancer].

PubMed

Akazawa, S; Harada, A; Futatsuki, K

1984-07-01

It is known that interstitial collagens are initially synthesized as precursors (procollagen), which possess extra peptide segments at both ends of the molecules. The authors attempted to detect the aminoterminal peptide of type III procollagen (type III-N-peptide) and also to measure the carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) together in sera of patients with gastric cancer. The results showed that: (1) mean serum levels and positive ratios of the type III-N-peptide increased as the clinical stage of the patients with gastric cancer advanced; (2) serum levels of the type III-N-peptide were not correlated either with those of CEA or CA 19-9; (3) positive ratios of type III-N-peptide, CEA and CA 19-9 were 51.7%, 44.8% and 48.3%, respectively: (4) positive ratio in combination of the type III-N-peptide with CEA was 69.3% and that in combination of the type III-N-peptide with CEA and CA 19-9 was 72.4%. These results suggest that type III-N-peptide is available for diagnosis of gastric cancer and, that the combination assay of type III-N-peptide with CEA and CA 19-9 is more effective than a single assay for diagnosis.

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

PubMed

Okano, Shinji; Matsumoto, Yoshihiro; Yoshiya, Shohei; Yamashita, Yo-ichi; Harimoto, Norifumi; Ikegami, Toru; Shirabe, Ken; Harada, Mamoru; Yoshikai, Yasunobu; Maehara, Yoshihiko

2013-01-01

The use of cancer immunotherapy as part of multidisciplinary therapies for cancer is a promising strategy for the cure of advanced cancer patients. In cancer immunotherapy, the effective priming of tumor-associated antigen (TAA)-specific CD8+ T cells is essential, and therefore, the appropriate selection of the best peptide for targeting the cancer is a most important concern. One criticism in the selection of a TAA is the immunogenicity of the TAA, the vaccination of which effectively elicits clinical responses. However, the critical basic immunological factors that affect the differences in the immunogenicity of TAAs remain to be elucidated. Here we found that CD4 T-cell responses suppressed the immunogenicity of the concomitant TAA in a murine melanoma model in which intratumoral activated dendritic therapy (ITADT) was used for treatment of the established cancer, and we observed that the antitumor effects were largely dependent on the CD8 T-cell response. CD4 T-cell depletion simply enhanced the tyrosinase-related protein (TRP)-2(180-188) peptide-specific cytotoxic T-cell (CTL) responses, and CD4 T-cell depletion provided immunogenicity for mgp100(25-33) peptide, to which a CTL response could not be detected at all in CD4 T-cell-intact mice in the early therapeutic phase. Further, the mgp100(25-33) peptide-specific CTL response again became undetectable after the recovery of CD4 T cells in previously CD4-depleted, tumor-eradicated mice, whereas the TRP-2(180-188) peptide-specific CTL response was still much stronger in CD4-depleted mice than in CD4-intact mice. These findings suggest that the CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

Cellular Pathway(S) of Antigen Processing and Presentation in Fish APC: Endosomal Involvement and Cell-Free Antigen Presentation

PubMed Central

Vallejo, Abbe N.; Miller, Norman W.; Harvey, Nancy E.; Cuchens, Marvin A.; Warr, Gregory W.

1992-01-01

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved. PMID:1343103

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for presentation of transformed self

PubMed Central

Mohammed, Fiyaz; Cobbold, Mark; Zarling, Angela L.; Salim, Mahboob; Barrett-Wilt, Gregory A.; Shabanowitz, Jeffrey; Hunt, Donald F.; Engelhard, Victor H.; Willcox, Benjamin E.

2008-01-01

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex (MHC) class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphorylated peptides on cancer cells provides an immunological signature of “transformed self”. Here, we demonstrate that phosphorylation can radically increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide–HLA-A2 complexes. These revealed a novel peptide binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting MHC binding, or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy. PMID:18836451

Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07.

PubMed

Marino, Fabio; Mommen, Geert P M; Jeko, Anita; Meiring, Hugo D; van Gaans-van den Brink, Jacqueline A M; Scheltema, Richard A; van Els, Cécile A C M; Heck, Albert J R

2017-01-06

Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy.

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.

PubMed

Padovan, E; Bauer, T; Tongio, M M; Kalbacher, H; Weltzien, H U

1997-06-01

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induced IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine epsilon-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.

Simulated digestion for testing the stability of edible vaccine based on Cucumber mosaic virus (CMV) chimeric particle display Hepatitis C virus (HCV) peptide.

PubMed

Vitti, Antonella; Nuzzaci, Maria; Condelli, Valentina; Piazzolla, Pasquale

2014-01-01

Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex.

PubMed

Elliott, Tim; Williams, Anthony

2005-10-01

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

Exosomal cancer immunotherapy is independent of MHC molecules on exosomes.

PubMed

Hiltbrunner, Stefanie; Larssen, Pia; Eldh, Maria; Martinez-Bravo, Maria-Jose; Wagner, Arnika K; Karlsson, Mikael C I; Gabrielsson, Susanne

2016-06-21

Peptide-loaded exosomes are promising cancer treatment vehicles; however, moderate T cell responses in human clinical trials indicate a need to further understand exosome-induced immunity. We previously demonstrated that antigen-loaded exosomes carry whole protein antigens and require B cells for inducing antigen-specific T cells. Therefore, we investigated the relative importance of exosomal major histocompatibility complex (MHC) class I for the induction of antigen-specific T cell responses and tumour protection. We show that ovalbumin-loaded dendritic cell-derived exosomes from MHCI-/- mice induce antigen-specific T cells at the same magnitude as wild type exosomes. Furthermore, exosomes lacking MHC class I, as well as exosomes with both MHC class I and II mismatch, induced tumour infiltrating T cells and increased overall survival to the same extent as syngeneic exosomes in B16 melanoma. In conclusion, T cell responses are independent of exosomal MHC/peptide complexes if whole antigen is present. This establishes the prospective of using impersonalised exosomes, and will greatly increase the feasibility of designing exosome-based vaccines or therapeutic approaches in humans.

Zoledronic acid induces dose-dependent increase of antigen-specific CD8 T-cell responses in combination with peptide/poly-IC vaccine.

PubMed

Park, Hye-Mi; Cho, Hyun-Il; Shin, Chang-Ae; Shon, Hyun-Jung; Kim, Tai-Gyu

2016-03-04

Zoledronic acid (ZA) is used for treating osteoporosis and for preventing skeletal fractures in cancer patients suffering from myeloma and prostate cancer. It is also reported to directly induce cancer cell apoptosis and indirectly modulate T-cell immune response as an antitumor agent. In this study, the effect of ZA following peptide/polyinosinic-polycytidylic acid (poly-IC) vaccination was investigated in a murine tumor model. The combination of ZA with peptide/poly-IC vaccine showed a synergistic effect on the induction of antigen-specific CD8 T-cell response. Three consecutive intravenous administrations of ZA was defined to induce the highest CD8 T-cell response. Further, total splenocyte counts and antigen-specific CD8 T-cell response gradually increased depending on the dose of ZA. In tumor-bearing mice, ZA showed a dose-dependent decrease of growth and prolonged survival. Treatment with ZA only decreased the number of CD11b(+)Gr1(+) myeloid cells in blood. Our results demonstrate that the use of ZA could improve antitumor immune responses induced by the peptide/poly-IC vaccine. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2013-06-01

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

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

PubMed

Sedlik, C; Dadaglio, G; Saron, M F; Deriaud, E; Rojas, M; Casal, S I; Leclerc, C

2000-07-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity†

PubMed Central

Sedlik, C.; Dadaglio, G.; Saron, M. F.; Deriaud, E.; Rojas, M.; Casal, S. I.; Leclerc, C.

2000-01-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+ T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies. PMID:10846055

Humoral immunity targeting site I of antigenic domain 2 of glycoprotein B upon immunization with different cytomegalovirus candidate vaccines.

PubMed

Axelsson, Fredrika; Adler, Stuart P; Lamarre, Alain; Ohlin, Mats

2007-12-21

Glycoprotein B (gB) is a major component in several vaccines that are under development for prevention of disease by cytomegalovirus. It contains multiple determinants that are targets for neutralizing antibodies. One of them is site I of antigenic domain 2 (AD-2). The epitope, defined by short peptides, is quite conserved between different isolates. However, it is poorly immunogenic in natural infection. In this study we investigated the extent to which different vaccines, attenuated live Towne vaccine with or without priming with a canarypox virus coding for gB, or a recombinant gB vaccine adjuvanted with MF59, induced antibodies to this epitope. As in natural infection only a fraction of all subjects developed antibody responses against site I of AD-2 following vaccination. We suggest that strategies that enhance immunogenicity of this epitope will improve vaccine efficacy.

Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease

PubMed Central

Panas, Michael W.; Mao, Rong; Delanoy, Michelle; Flanagan, John J.; Binder, Steven R.; Rebman, Alison W.; Montoya, Jose G.; Soloski, Mark J.; Steere, Allen C.; Dattwyler, Raymond J.; Arnaboldi, Paul M.; Aucott, John N.

2015-01-01

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease. PMID:26447113

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

PubMed

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

2008-11-01

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

The Control of the Specificity of CD4 T Cell Responses: Thresholds, Breakpoints, and Ceilings

PubMed Central

Sant, Andrea J.; Chaves, Francisco A.; Leddon, Scott A.; Tung, Jacqueline

2013-01-01

It has been known for over 25 years that CD4 T cell responses are restricted to a finite number of peptide epitopes within pathogens or protein vaccines. These selected peptide epitopes are termed “immunodominant.” Other peptides within the antigen that can bind to host MHC molecules and recruit CD4 T cells as single peptides are termed “cryptic” because they fail to induce responses when expressed in complex proteins or when in competition with other peptides during the immune response. In the last decade, our laboratory has evaluated the mechanisms that underlie the preferential specificity of CD4 T cells and have discovered that both intracellular events within antigen presenting cells, particular selective DM editing, and intercellular regulatory pathways, involving IFN-γ, indoleamine 2,3-dioxygenase, and regulatory T cells, play a role in selecting the final peptide specificity of CD4 T cells. In this review, we summarize our findings, discuss the implications of this work on responses to pathogens and vaccines and speculate on the logic of these regulatory events. PMID:24167504

Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

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

McLellan, Jason S.; Yang, Yongping; Graham, Barney S.

2011-09-16

Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in itsmore » postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.« less

An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes.

PubMed

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI(+) colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. FSP-specific CD8(+) T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI(+) colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were (386)-FLLALWECSL (FSP18) and (387)-LLALWECSL (FSP19) as well as (403)-IVSRTLLLV (FSP23) and (402)-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI(+)-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or--even more important--preventive purposes.

Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for efficient CD8(+) T cell activation.

PubMed

Silva, A L; Rosalia, R A; Sazak, A; Carstens, M G; Ossendorp, F; Oostendorp, J; Jiskoot, W

2013-04-01

Overlapping synthetic long peptides (SLPs) hold great promise for immunotherapy of cancer. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are being developed as delivery systems to improve the potency of peptide-based therapeutic cancer vaccines. Our aim was to optimize PLGA NP for SLP delivery with respect to encapsulation and release, using OVA24, a 24-residue long synthetic antigenic peptide covering a CTL epitope of ovalbumin (SIINFEKL), as a model antigen. Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique. Using standard conditions (acidic inner aqueous phase), we observed that either encapsulation was very low (1-30%), or burst release extremely high (>70%) upon resuspension of NP in physiological buffers. By adjusting formulation and process parameters, we uncovered that the pH of the first emulsion was critical to efficient encapsulation and controlled release. In particular, an alkaline inner aqueous phase resulted in circa 330 nm sized NP with approximately 40% encapsulation efficiency and low (<10%) burst release. These NP showed enhanced MHC class I restricted T cell activation in vitro when compared to high-burst releasing NP and soluble OVA24, proving that efficient entrapment of the antigen is crucial to induce a potent cellular immune response. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacteriophage-Fused Peptides for Serodiagnosis of Human Strongyloidiasis

PubMed Central

Feliciano, Nágilla Daliane; Ribeiro, Vanessa da Silva; Santos, Fabiana de Almeida Araújo; Fujimura, Patricia Tiemi; Gonzaga, Henrique Tomaz; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

2014-01-01

Background Strongyloidiasis, a human intestinal infection caused by the nematode Strongyloides stercoralis, is frequently underdiagnosed and although its high prevalence is still a neglected parasitic disease because conventional diagnostic tests based on parasitological examination (presence of Strongyloides larvae in stool) are not sufficiently sensitive due to the low parasitic load and to the irregular larval output. There is an urgent need to improve diagnostic assays, especially for immunocompromised patients with high parasitic load as consequence of self-infection cycle, which can disseminate throughout the body, resulting in a potentially fatal hyperinfection syndrome often accompanied by sepsis or meningitis. Methods/Principal Findings We have performed Phage Display technology to select peptides that mimic S. stercoralis antigens, capable of detecting a humoral response in patients with strongyloidiasis. The peptides reactivity was investigated by Phage-ELISA through different panels of serum samples. We have successfully selected five peptides with significant immunoreactivity to circulating IgG from patients' sera with strongyloidiasis. The phage displayed peptides C9 and C10 presented the highest diagnostic potential (AUC>0.87) with excellent sensitivity (>85%) and good specificity (>77.5%), suggesting that some S. stercoralis antigens trigger systemic immune response. Conclusions/Significance These novel antigens are interesting serum biomarkers for routine strongyloidiasis screenings due to the easy production and simple assay using Phage-ELISA. Such markers may also present a promising application for therapeutic monitoring. PMID:24874206

Minimally Activated CD8 Autoreactive T Cells Specific for IRBP Express a High Level of Foxp3 and Are Functionally Suppressive

PubMed Central

Peng, Yong; Shao, Hui; Ke, Yan; Zhang, Ping; Han, Gencheng; Kaplan, Henry J.; Sun, Deming

2008-01-01

Purpose Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide. Methods Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retin-oid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity. Results CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 μg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-β1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function. Conclusions Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells. PMID:17460277

Engineered hybrid spider silk particles as delivery system for peptide vaccines.

PubMed

Lucke, Matthias; Mottas, Inès; Herbst, Tina; Hotz, Christian; Römer, Lin; Schierling, Martina; Herold, Heike M; Slotta, Ute; Spinetti, Thibaud; Scheibel, Thomas; Winter, Gerhard; Bourquin, Carole; Engert, Julia

2018-07-01

The generation of strong T-cell immunity is one of the main challenges for the development of successful vaccines against cancer and major infectious diseases. Here we have engineered spider silk particles as delivery system for a peptide-based vaccination that leads to effective priming of cytotoxic T-cells. The recombinant spider silk protein eADF4(C16) was fused to the antigenic peptide from ovalbumin, either without linker or with a cathepsin cleavable peptide linker. Particles prepared from the hybrid proteins were taken up by dendritic cells, which are essential for T-cell priming, and successfully activated cytotoxic T-cells, without signs of immunotoxicity or unspecific immunostimulatory activity. Upon subcutaneous injection in mice, the particles were taken up by dendritic cells and accumulated in the lymph nodes, where immune responses are generated. Particles from hybrid proteins containing a cathepsin-cleavable linker induced a strong antigen-specific proliferation of cytotoxic T-cells in vivo, even in the absence of a vaccine adjuvant. We thus demonstrate the efficacy of a new vaccine strategy using a protein-based all-in-one vaccination system, where spider silk particles serve as carriers with an incorporated peptide antigen. Our study further suggests that engineered spider silk-based vaccines are extremely stable, easy to manufacture, and readily customizable. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+ APC and antigen-specific T cell responses in vitro.

PubMed

Gogesch, Patricia; Schülke, Stefan; Scheurer, Stephan; Mühlebach, Michael D; Waibler, Zoe

2018-05-28

The development of novel vaccination strategies is a persistent challenge to provide effective prophylactic treatments to encounter viral infections. In general, the physical conjugation of selected vaccine components, e.g. antigen and adjuvant, has been shown to enhance the immunogenicity and hence, can increase effectiveness of the vaccine. In our proof-of-concept study, we generated non-infectious, replication deficient Murine Leukemia Virus (MLV)-derived virus-like particles (VLPs) that physically link antigen and adjuvant in a modular fashion by co-displaying them on their surface. For this purpose, we selected the immunodominant peptides of the model antigen ovalbumin (OVA) and the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) as non-classical adjuvant. Our results show that murine GM-CSF displayed on MLV-VLPs mediates expansion and proliferation of CD11b + cells within murine bone marrow and total spleen cells. Moreover, we show increased immunogenicity of modular VLPs co-displaying OVA peptides and GM-CSF by their elevated capacity to induce OVA-specific T cell-activation and -proliferation within OT-I and OT-II splenocyte cultures. These enhanced effects were not achieved by using an equimolar mixture of VLPs displaying either OVA or GM-CSF. Taken together, OVA and GM-CSF co-displaying MLV-VLPs are able to target and expand antigen presenting cells which in turn results in enhanced antigen-specific T cell activation and proliferation in vitro. These data suggest MLV-VLPs to be an attractive platform to flexibly combine antigen and adjuvant for novel modular vaccination approaches. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

PubMed

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

2003-11-07

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

Designing peptide inhibitor of insulin receptor to induce diabetes mellitus type 2 in animal model Mus musculus.

PubMed

Permatasari, Galuh W; Utomo, Didik H; Widodo

2016-10-01

A designing peptide as agent for inducing diabetes mellitus type 2 (T2DM) in an animal model is challenging. The computational approach provides a sophisticated tool to design a functional peptide that may block the insulin receptor activity. The peptide that able to inhibit the binding between insulin and insulin receptor is a warrant for inducing T2DM. Therefore, we designed a potential peptide inhibitor of insulin receptor as an agent to generate T2DM animal model by bioinformatics approach. The peptide has been developed based on the structure of insulin receptor binding site of insulin and then modified it to obtain the best properties of half life, hydrophobicity, antigenicity, and stability binding into insulin receptor. The results showed that the modified peptide has characteristics 100h half-life, high-affinity -95.1±20, and high stability 28.17 in complex with the insulin receptor. Moreover, the modified peptide has molecular weight 4420.8g/Mol and has no antigenic regions. Based on the molecular dynamic simulation, the complex of modified peptide-insulin receptor is more stable than the commercial insulin receptor blocker. This study suggested that the modified peptide has the promising performance to block the insulin receptor activity that potentially induce diabetes mellitus type 2 in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis1

PubMed Central

Whitaker, Paul; Meng, Xiaoli; Lavergne, Sidonie N.; El-Ghaiesh, Sabah; Monshi, Manal; Earnshaw, Caroline; Peckham, Daniel; Gooi, Jimmy; Conway, Steve; Pirmohamed, Munir; Jenkins, Rosalind E.; Naisbitt, Dean J.; Park, B. Kevin

2011-01-01

A mechanistic understanding of the relationship between the chemistry of drug antigen formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating antigens derived from piperacillin in patients undergoing therapy and the nature of the drug derived-epitopes on protein which can function as an antigen to stimulate T-cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time- and concentration-dependent, with selective modification of Lys541 observed at low concentrations, whereas at higher concentrations up to 13/59 lysine residues were modified, four of which (Lys190, 195, 432 and 541) were detected in patients’ plasma. Piperacillin-specific T-lymphocyte responses (proliferation, cytokines and granzyme-B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T-cells with characterized synthetic conjugates. Analysis of minimally-modified T-cell stimulatory albumin conjugates revealed peptide sequences incorporating Lys190, 432 and 541 as principal functional epitopes for T-cells. This study has characterized the multiple haptenic structures on albumin in patients, and showed that they constitute functional antigenic determinants for T-cells. PMID:21606251

Development of a Peptide ELISA for the Diagnosis of Aleutian Mink Disease

PubMed Central

Wang, Yang; Lu, Rongguang; Hu, Bo; Lv, Shuang; Xue, Xianghong; Li, Xintong; Ling, Mingyu; Fan, Sining; Zhang, Hailing; Yan, Xijun

2016-01-01

Aleutian disease (AD) is a common immunosuppressive disease in mink farms world-wide. Since the 1980s, counterimmunoelectrophoresis (CIEP) has been the main detection method for infection with the Aleutian Mink Disease Virus (AMDV). In this study, six peptides derived from the AMDV structural protein VP2 were designed, synthesized, and used as ELISA antigens to detect anti-AMDV antibodies in the sera of infected minks. Serum samples were collected from 764 minks in farms from five different provinces, and analyzed by both CIEP (a gold standard) and peptide ELISA. A peptide designated P1 (415 aa–433 aa) exhibited good antigenicity. A novel ELISA was developed using ovalbumin-linked peptide P1 to detect anti-AMDV antibodies in mink sera. The sensitivity and specificity of the peptide ELISA was 98.0% and 97.5%, respectively. Moreover, the ELISA also detected 342 early-stage infected samples (negative by CIEP and positive by PCR), of which 43.6% (149/342) were true positives. These results showed that the peptide ELISA had better sensitivity compared with CIEP, and therefore could be preferable over CIEP for detecting anti-AMDV antibodies in serological screening. PMID:27802320

Chimeric peptide containing both B and T cells epitope of tumor-associated antigen L6 enhances anti-tumor effects in HLA-A2 transgenic mice.

PubMed

Lin, Su-I; Huang, Ming-Hsi; Chang, Yu-Wen; Chen, I-Hua; Roffler, Steve; Chen, Bing-Mae; Sher, Yuh-Pyng; Liu, Shih-Jen

2016-07-28

Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A T-Cell Receptor Breaks the Rules | Center for Cancer Research

Cancer.gov

Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their parents; and the antigen fragment, called a peptide epitope, is excised from one of thousands of possible proteins—originally part of an invading pathogen or a cancer cell—that T cells are capable of identifying and attacking. The framework of an MHC molecule holding a centrally displayed or “presented” peptide is what engages the TCR and triggers T-cell action. This role of MHC molecules presenting antigens to the TCR is a central tenet of immunology, with the fit between a TCR and the MHC framework actually “hardwired” into their three-dimensional structures.

Co-delivery of antigen and a lipophilic anti-inflammatory drug to cells via a tailorable nanocarrier emulsion.

PubMed

Chuan, Yap Pang; Zeng, Bi Yun; O'Sullivan, Brendan; Thomas, Ranjeny; Middelberg, Anton P J

2012-02-15

Nanotechnology promises new drug carriers that can be tailored to specific applications. Here we report a new approach to drug delivery based on tailorable nanocarrier emulsions (TNEs), motivated by a need to co-deliver a protein antigen and a lipophilic drug for specific inhibition of nuclear factor kappa B (NF-κB) in antigen presenting cells (APCs). Co-delivery for NF-κB inhibition holds promise as a strategy for the treatment of rheumatoid arthritis. We used a highly surface-active peptide (SAP) to prepare a nanosized emulsion having defined surface properties predictable from the SAP sequence. Incorporating the lipophilic drug into the oil phase at the time of emulsion formation enabled its facile packaging. The SAP is depleted from bulk during emulsification, allowing simple subsequent addition of the drug-loaded oil-in-water emulsion to a solution of protein antigen. Decoration of emulsion surface with antigen was achieved via electrostatic deposition. In vitro data showed that the TNE prepared this way was internalized and well-tolerated by model APCs, and that good suppression of NF-κB expression was achieved. This work reports a new type of nanotechnology-based carrier, a TNE, which can potentially be tailored for co-delivery of multiple therapeutic components, and can be made using simple methods using only biocompatible materials. Copyright © 2011 Elsevier Inc. All rights reserved.

Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs.

PubMed

Hiremath, Jagadish; Kang, Kyung-il; Xia, Ming; Elaish, Mohamed; Binjawadagi, Basavaraj; Ouyang, Kang; Dhakal, Santosh; Arcos, Jesus; Torrelles, Jordi B; Jiang, X; Lee, Chang Won; Renukaradhya, Gourapura J

2016-01-01

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove

PubMed Central

Rohrer, Karin M; Haug, Markus; Schwörer, Daniela; Kalbacher, Hubert; Holzer, Ursula

2014-01-01

Heat-shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I-and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously. PMID:24428437

The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease

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

Henderson, Kate N.; Reid, Hugh H.; Borg, Natalie A.

2007-12-01

The production and crystallization of human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 in complex with deamidated gliadin peptides is reported. Crystals of HLA-DQ2{sup PQPELPYPQ} diffracted to 3.9 Å, while the HLA-DQ8{sup EGSFQPSQE} crystals diffracted to 2.1 Å, allowing structure determination by molecular replacement. The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4{sup +} T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 α-I (PQPELPYPQ) and DQ8more » α-I (EGSFQPSQE), respectively, are reported.« less

High-Density Peptide Arrays for Malaria Vaccine Development.

PubMed

Loeffler, Felix F; Pfeil, Johannes; Heiss, Kirsten

2016-01-01

The development of an efficacious and practicable vaccine conferring sterile immunity towards a Plasmodium infection represents a not yet achieved goal. A crucial factor for the impact of a given anti-plasmodial subunit vaccine is the identification of the most potent parasitic components required to induce protection from both infection and disease. Here, we present a method based on a novel high-density peptide array technology that allows for a flexible readout of malaria antibodies. Peptide arrays applied as a screening method can be used to identify novel immunogenic antibody epitopes under a large number of potential antigens/peptides. Ultimately, discovered antigen candidates and/or epitope sequences can be translated into vaccine prototype design. The technology can be further utilized to unravel antibody-mediated immune responses (e.g., involved in the establishment of semi-immunity) and moreover to confirm vaccine potency during the process of clinical development by verifying the induced antibody responses following vaccination.

Structural Elements Recognized by Abacavir-Induced T Cells

PubMed Central

Yerly, Daniel; Pompeu, Yuri Andreiw; Schutte, Ryan J.; Eriksson, Klara. K.; Strhyn, Anette; Bracey, Austin. W.; Buus, Soren; Ostrov, David A.

2017-01-01

Adverse drug reactions are one of the leading causes of morbidity and mortality in health care worldwide. Human leukocyte antigen (HLA) alleles have been strongly associated with drug hypersensitivities, and the causative drugs have been shown to stimulate specific T cells at the sites of autoimmune destruction. The structural elements recognized by drug-specific T cell receptors (TCRs) in vivo are poorly defined. Drug-stimulated T cells express TCRs specific for peptide/HLA complexes, but the characteristics of peptides (sequence, or endogenous or exogenous origin) presented in the context of small molecule drugs are not well studied. Using HLA-B*57:01 mediated hypersensitivity to abacavir as a model system, this study examines structural similarities of HLA presented peptides recognized by drug-specific TCRs. Using the crystal structure of HLA-B*57:01 complexed with abacavir and an immunogenic self peptide, VTTDIQVKV SPT5a 976–984, peptide side chains exhibiting flexibility and solvent exposure were identified as potential drug-specific T cell recognition motifs. Viral sequences with structural motifs similar to the immunogenic self peptide were identified. Abacavir-specific T cell clones were used to determine if virus peptides presented in the context of abacavir stimulate T cell responsiveness. An abacavir-specific T cell clone was stimulated by VTQQAQVRL, corresponding to HSV1/2 230–238, in the context of HLA-B*57:01. These data suggest the T cell polyclonal response to abacavir consists of multiple subsets, including T cells that recognize self peptide/HLA-B*57:01 complexes and crossreact with viral peptide/HLA-B*57:01 complexes due to similarity in TCR contact residues. PMID:28686208

Peptide probes reveal a hydrophobic steric ratchet in the anthrax toxin protective antigen translocase

PubMed Central

Colby, Jennifer M.; Krantz, Bryan A.

2015-01-01

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor (LF) and edema factor (EF), into the host cytosol under the proton motive force (PMF). Protein translocation under a PMF is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between LF amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides; and while these steric interactions may make a peptide translocate poorly, in the context of folded domains they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism. PMID:26363343

Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen.

PubMed

Sandomenico, Annamaria; Focà, Annalia; Sanguigno, Luca; Caporale, Andrea; Focà, Giuseppina; Pignalosa, Angelica; Corvino, Giusy; Caragnano, Angela; Beltrami, Antonio Paolo; Antoniali, Giulia; Tell, Gianluca; Leonardi, Antonio; Ruvo, Menotti

Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24-39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.

Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data.

PubMed

Schittenhelm, Ralf B; Sivaneswaran, Saranjah; Lim Kam Sian, Terry C C; Croft, Nathan P; Purcell, Anthony W

2016-06-01

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

PubMed Central

Wieczorek, Marek; Abualrous, Esam T.; Sticht, Jana; Álvaro-Benito, Miguel; Stolzenberg, Sebastian; Noé, Frank; Freund, Christian

2017-01-01

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

Complex pattern of immune evasion in MSI colorectal cancer.

PubMed

Ozcan, Mine; Janikovits, Jonas; von Knebel Doeberitz, Magnus; Kloor, Matthias

2018-01-01

Mismatch repair (MMR)-deficient cancers accumulate multiple insertion/deletion mutations at coding microsatellites (cMS), which give rise to frameshift peptide neoantigens. The high mutational neoantigen load of MMR-deficient cancers is reflected by pronounced anti-tumoral immune responses of the host and high responsiveness towards immune checkpoint blockade. However, immune evasion mechanisms can interfere with the immune response against MMR-deficient tumors. We here performed a comprehensive analysis of immune evasion in MMR-deficient colorectal cancers, focusing on HLA class I-mediated antigen presentation. 72% of MMR-deficient colorectal cancers of the DFCI database harbored alterations affecting genes involved in HLA class I-mediated antigen presentation, and 54% of these mutations were predicted to abrogate function. Mutations affecting the HLA class I transactivator NLRC5 were observed as a potential new immune evasion mechanism in 26% (6% abrogating) of the analyzed tumors. NLRC5 mutations in MMR-deficient cancers were associated with decreased levels of HLA class I antigen expression. In summary, the majority of MMR-deficient cancers display mutations interfering with HLA class I antigen presentation that reflect active immune surveillance and immunoselection during tumor development. Clinical studies focusing on immune checkpoint blockade in MSI cancer should account for the broad variety of immune evasion mechanisms as potential biomarkers of therapy success.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

PubMed

Kessler, J H; Bres-Vloemans, S A; van Veelen, P A; de Ru, A; Huijbers, I J G; Camps, M; Mulder, A; Offringa, R; Drijfhout, J W; Leeksma, O C; Ossendorp, F; Melief, C J M

2006-10-01

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Papac, D I; Hoyes, J; Tomer, K B

1994-09-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed Central

Papac, D. I.; Hoyes, J.; Tomer, K. B.

1994-01-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID:7530543

Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

PubMed

Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

2016-02-29

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines.

PubMed

Rauta, Pradipta R; Nayak, Bismita; Monteiro, Gabriel A; Mateus, Marília

2017-01-10

The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector. The selected DNA vaccine candidates were purified from E. coli DH5α and transfected into Chinese hamster ovary cells. The expression of the antigenic peptides was measured in cells along post-transfection time, through the fluorescence intensity of the reporter GFP. The lipofection efficiency of aha-pVAX-GFP was highest after 24h incubation. Formulated PLGA-chitosan nanoparticle/plasmid DNA complexes were characterized in terms of size, size distribution and zeta potential. Nanocomplexes with average diameters in the range of 150-170nm transfected in a similar fashion into CHO cells confirmed transfection efficiency comparable to that of lipofection. DNA entrapment and further DNase digestion assays demonstrated ability for pDNA protection by the nanoparticles against enzymatic digestion. Copyright © 2016 Elsevier B.V. All rights reserved.

Perspectives for immunotherapy in glioblastoma treatment.

PubMed

Finocchiaro, Gaetano; Pellegatta, Serena

2014-11-01

Avoiding immune destruction is one emerging hallmark of cancer, including glioblastoma. The number of immunotherapy approaches to fight glioblastoma is growing. Here, we review the recent progress in four main areas: dendritic cell immunotherapy, peptide vaccination, chimeric antigen receptors and immune checkpoints. We and others are using dendritic cells to present glioblastoma antigens (whole tumor lysate) to the immune system; our initial data indicate that clinical benefit is associated to increased presence of natural killer cells in the periphery. A pilot study loading dendritic cells with glioblastoma stem-like cells will start soon. Peptide vaccination targeting the epidermal growth factor receptor variant III (EGFRvIII) epitope, present in 25% of glioblastomas, is ongoing. Intriguing results have been obtained by vaccination with three other peptides in pediatric gliomas. Another clinical trial is targeting EGFRvIII by adoptive cell transfer of chimeric antigen receptor. This exciting technology could be suited for a number of other potential epitopes discovered through next-generation sequencing. Finally, antibodies against the immune checkpoints cytotoxic T lymphocyte antigen-4 and programmed cell death-1, which demonstrated efficacy in advanced melanomas, will be used in novel trials for recurrent glioblastoma. In all these studies attention to novel side-effects and to MRI as immunological follow-up to distinguish progression or pseudoprogression will be of critical relevance.

Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum.

PubMed

Ayela, Cedric; Roquet, Francoise; Valera, Lionel; Granier, Claude; Nicu, Liviu; Pugnière, Martine

2007-06-15

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG(6)) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2 nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.

Characterization and serology of the matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni before and after degradation by an endogenous proteinase.

PubMed

Eppstein, D A; Thoma, J A

1977-11-01

The intact matrix protein from a nuclear-polyhedrosis virus of the cabbage looper (Trichoplusia ni), isolated after inhibition of an endogenous serine-type proteinase, was further purified by molecular-sieve chromatography. The matrix protein was associated with carbohydrate moieties, and the carbohydrate content was determined for the two major peptides isolated after proteolysis by the endogenous proteinase. The association-dissociation interactions of the intact and proteinase-hydrolysed monomer units were characterized at high and low pH. At pH1.9, proteinase-degraded matrix protein dissociated into two different peptide fractions, FI and FII. Fraction FII, a single peptide of 9400 daltons, comprised one-third of the monomer unit of 28 000 daltons. At pH9.5, the degraded peptides were tightly associated in units equivalent to the intact monomer. These monomer equivalents associated to form a series of interconverting aggregates. The predominant aggregate sedimented at 11S and had a mol.wt greater than or equal to 200 000. Two non-cross-reacting antigens were present in the aggregate mixture. The presence of these two antigens does not reflect the presence of two different matrix proteins; rather, the expression of the antigens correlates with the degree of aggregation of the matrix protein.

Regions of botulinum neurotoxin A light chain recognized by human anti-toxin antibodies from cervical dystonia patients immunoresistant to toxin treatment. The antigenic structure of the active toxin recognized by human antibodies.

PubMed

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

2011-07-01

This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141-159), L14 (183-201) and L18 (239-257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365-383) and L29 (379-397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation. Copyright © 2011 Elsevier GmbH. All rights reserved.

Auto-inhibition and phosphorylation-induced activation of PLC-γ isozymes

PubMed Central

Hajicek, Nicole; Charpentier, Thomas H.; Rush, Jeremy R.; Harden, T. Kendall; Sondek, John

2013-01-01

Multiple extracellular stimuli, such as growth factors and antigens, initiate signaling cascades through tyrosine phosphorylation and activation of phospholipase C (PLC)-γ isozymes. Like most other PLCs, PLC-γ1 is basally auto-inhibited by its X-Y linker, which separates the X-and Y-boxes of the catalytic core. The C-terminal SH2 (cSH2) domain within the X-Y linker is the critical determinant for auto-inhibition of phospholipase activity. Release of auto-inhibition requires an intramolecular interaction between the cSH2 domain and a phosphorylated tyrosine, Tyr783, also located within the X-Y linker. The molecular mechanisms that mediate auto-inhibition and phosphorylation-induced activation have not been defined. Here, we describe structures of the cSH2 domain both alone and bound to a PLC-γ1 peptide encompassing phosphorylated Tyr783. The cSH2 domain remains largely unaltered by peptide engagement. Point mutations in the cSH2 domain located at the interface with the peptide were sufficient to constitutively activate PLC-γ1 suggesting that peptide engagement directly interferes with the capacity of the cSH2 domain to block the lipase active site. This idea is supported by mutations in a complimentary surface of the catalytic core that also enhanced phospholipase activity. PMID:23777354

A role for mitochondria in antigen processing and presentation

PubMed Central

Bonifaz, Laura C; Cervantes-Silva, Mariana P; Ontiveros-Dotor, Elizabeth; López-Villegas, Edgar O; Sánchez-García, F Javier

2015-01-01

Immune synapse formation is critical for T-lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen-presenting cells (APCs) and T lymphocytes is a two-way signalling process. However, the role of mitochondria in APCs during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing and -presentation process. Here we show that hen egg white lysozyme (HEL) -loaded B lymphocytes, as a type of APC, undergo a small but significant mitochondrial depolarization by 1–2 hr following antigen exposure, suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca2+ uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analysed. Oligomycin treatment reduced the amount of specific MHC–peptide complexes but not total MHC II on the cell membrane of B lymphocytes, which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes, which endogenously express HEL and by B lymphocytes loaded with the HEL48–62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taken together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APC mitochondria were found to re-organize towards the APC–T immune synapse. PMID:25251370

77 FR 26014 - Prospective Grant of Exclusive License: P4 Peptide From Streptococcus Pneumoniae

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-02

... ``Functional Epitopes of Streptococcus Pneumoniae PsaA Antigen and Uses Thereof,'' filed 7/ 18/2008, claiming.... Technology This technology consists of a P4 peptide which contains functional epitopes of the PsaA protein of...

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

PubMed Central

Ahn, K; Meyer, T H; Uebel, S; Sempé, P; Djaballah, H; Yang, Y; Peterson, P A; Früh, K; Tampé, R

1996-01-01

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP. Images PMID:8670825

A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides

PubMed Central

1994-01-01

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules. PMID:8294869

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

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

Brodzik, R.; Bandurska, K.; Deka, D.

2005-12-16

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles weremore » efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.« less

Carbohydrates and T cells: A sweet twosome

PubMed Central

Avci, Fikri Y.; Li, Xiangming; Tsuji, Moriya; Kasper, Dennis L.

2013-01-01

Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically-linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease. PMID:23757291

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

PubMed

Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza

2017-07-01

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

Atassi, M Z

2015-12-01

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

Fine specificity of antigen binding to two class I major histocompatibility proteins (B*2705 and B*2703) differing in a single amino acid residue

NASA Astrophysics Data System (ADS)

Rognan, Didier; Krebs, Stefan; Kuonen, Oliver; Lamas, , José R.; Castro, José A. López de; Folkers, Gerd

1997-09-01

Starting from the X-ray structure of a class I majorhistocompatibility complex (MHC)-encoded protein (HLA-B*2705), a naturallypresented self-nonapeptide and two synthetic analogues were simulated in thebinding groove of two human leukocyte antigen (HLA) alleles (B*2703 andB*2705) differing in a single amino acid residue. After 200 ps moleculardynamics simulations of the solvated HLA-peptide pairs, some molecularproperties of the complexes (distances between ligand and protein center ofmasses, atomic fluctuations, buried versus accessible surface areas,hydrogen-bond frequencies) allow a clear discrimination of potent from weakMHC binders. The binding specificity of the three nonapeptides for the twoHLA alleles could be explained by the disruption of one hydrogen-bondingnetwork in the binding pocket of the HLA-B*2705 protein where the singlemutation occurs. Rearrangements of interactions in the B pocket, which bindsthe side chain of peptidic residue 2, and a weakening of interactionsinvolving the C-terminal end of the peptide also took place. In addition,extension of the peptide backbone using a β-Ala analogue did notabolish binding to any of the two HLA-B27 subtypes, but increased theselectivity for B*2703, as expected from the larger peptide binding groovein this subtype. A better understanding of the atomic details involved inpeptide selection by closely related HLA alleles is of crucial importancefor unraveling the molecular features linking particular HLA alleles toautoimmune diseases, and for the identification of antigenic peptidestriggering such pathologies.

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

PubMed

Takeda, Kazuyoshi; Kitaura, Kazutaka; Suzuki, Ryuji; Owada, Yuki; Muto, Satoshi; Okabe, Naoyuki; Hasegawa, Takeo; Osugi, Jun; Hoshino, Mika; Tsunoda, Takuya; Okumura, Ko; Suzuki, Hiroyuki

2018-06-01

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca 2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

PubMed Central

Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

1990-01-01

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

Mapping the tumour human leukocyte antigen (HLA) ligandome by mass spectrometry.

PubMed

Freudenmann, Lena Katharina; Marcu, Ana; Stevanović, Stefan

2018-07-01

The entirety of human leukocyte antigen (HLA)-presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide-specific immunotherapies, MS-based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution, and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and deconvolute multi-allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA-presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide-specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS-guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application, and provide an outlook on future developments. © 2018 John Wiley & Sons Ltd.

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

PubMed Central

1992-01-01

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

Immunocontraceptive efficacy of synthetic peptides corresponding to major antigenic determinants of chicken riboflavin carrier protein in the female rats.

PubMed

Subramanian, S; Karande, A A; Adiga, P R

2000-09-01

Earlier studies have demonstrated that antibodies directed towards the N-terminal (residues 10-17) and C-terminal (residues 200-207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub-human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33-49, 64 83 and 130-147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated. The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies. All the three peptides-specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE-IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy. These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini-vaccines.

PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.

PubMed

Chen, Wenhan; Guo, William W; Huang, Yanxin; Ma, Zhiqiang

2012-01-01

Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/

A B Cell-Driven Autoimmune Pathway Leading to Pathological Hallmarks of Progressive Multiple Sclerosis in the Marmoset Experimental Autoimmune Encephalomyelitis Model

PubMed Central

’t Hart, Bert A.; Dunham, Jordon; Faber, Bart W.; Laman, Jon D.; van Horssen, Jack; Bauer, Jan; Kap, Yolanda S.

2017-01-01

The absence of pathological hallmarks of progressive multiple sclerosis (MS) in commonly used rodent models of experimental autoimmune encephalomyelitis (EAE) hinders the development of adequate treatments for progressive disease. Work reviewed here shows that such hallmarks are present in the EAE model in marmoset monkeys (Callithrix jacchus). The minimal requirement for induction of progressive MS pathology is immunization with a synthetic peptide representing residues 34–56 from human myelin oligodendrocyte glycoprotein (MOG) formulated with a mineral oil [incomplete Freund’s adjuvant (IFA)]. Pathological aspects include demyelination of cortical gray matter with microglia activation, oxidative stress, and redistribution of iron. When the peptide is formulated in complete Freund’s adjuvant, which contains mycobacteria that relay strong activation signals to myeloid cells, oxidative damage pathways are strongly boosted leading to more intensive pathology. The proven absence of immune potentiating danger signals in the MOG34–56/IFA formulation implies that a narrow population of antigen-experienced T cells present in the monkey’s immune repertoire is activated. This novel pathway involves the interplay of lymphocryptovirus-infected B cells with MHC class Ib/Caja-E restricted CD8+ CD56+ cytotoxic T lymphocytes. PMID:28744286

Liposomes containing NY‑ESO‑1/tetanus toxoid and adjuvant peptides targeted to human dendritic cells via the Fc receptor for cancer vaccines.

PubMed

Cruz, Luis J; Rueda, Felix; Simón, Lorena; Cordobilla, Begoña; Albericio, Fernando; Domingo, Joan C

2014-04-01

To improve the immunological response against tumors, a vaccine based on nanoliposomes targeted to the Fcg-receptor was developed to enhance the immunogenicity of tumor-associated antigens (TAAs). Using human dendritic cells in vitro, a fragment of the TAA NY-ESO-1 combined with a T-helper peptide from the tetanus toxoid encapsulated in nanoliposomes was evaluated. In addition, peptides Palm-IL-1 and MAP-IFN-g were coadministered as adjuvants to enhance the immunological response. Coadministration of Palm-IL-1 or MAP-IFN-g peptide adjuvants and the hybrid NY-ESO-1-tetanus toxoid (soluble or encapsulated in nanoliposomes without targeting) increased immunogenicity. However, the most potent immunological response was obtained when the peptide adjuvants were encapsulated in liposomes targeted to human dendritic cells via the Fc receptor. This targeted vaccine strategy is a promising tool to activate and deliver antigens to dendritic cells, thus improving immunotherapeutic response in situations in which the immune system is frequently compromised, as in advanced cancers.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

PubMed

Okochi, Mina; Muto, Masaki; Yanai, Kentaro; Tanaka, Masayoshi; Onodera, Takeshi; Wang, Jin; Ueda, Hiroshi; Toko, Kiyoshi

2017-10-09

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single-cell level

NASA Astrophysics Data System (ADS)

Das, Gobind; La Rocca, Rosanna; Lakshmikanth, Tadepally; Gentile, Francesco; Tallerico, Rossana; Zambetti, Lia P.; Devitt, J.; Candeloro, Patrizio; de Angelis, Francesco; Carbone, Ennio; di Fabrizio, Enzo

2010-03-01

Human leukocyte antigen (HLA) class I molecules are formed by three immunoglobulin-like domains (α1, α2, and α3) once folded by peptide and β2-microglobulin show the presence of two α-helix streams and one β-sheet limiting the pocket for the antigenic peptide. The loss of HLA class I expression in tumors and virus-infected cells, on one hand, prevents T cell recognition, while on the other hand, it leads to natural killer (NK) cell mediated cytotoxicity. We propose the possibility of using Raman spectroscopy to measure the relative expression of HLA class I molecules at the single-cell level. Raman spectra are recorded for three cell lines (K562, T2, and T3) and monomers (HLA class I folded, unfolded and peptide+β2-microlobulin refolded) using 830 nm laser line. Our data are consistent with the hypothesis that in the Raman spectra, ranging from 1600 to 1800 cm-1, the intensity variation of cells associated with HLA class I molecules could be measured.

Expression of the gastrin-releasing peptide receptor, the prostate stem cell antigen and the prostate-specific membrane antigen in lymph node and bone metastases of prostate cancer.

PubMed

Ananias, Hildo J K; van den Heuvel, Marius C; Helfrich, Wijnand; de Jong, Igle J

2009-07-01

Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we investigated in this study whether these antigens are expressed in metastasized prostate cancer. Formalin-fixed, paraffin-embedded specimens of 15 patients with uni- or bilateral lymph node metastases of prostate cancer (totaling 21 cases) and 17 patient-cases of bone metastases were processed for immunohistochemistry with anti-GRPR, anti-PSCA, and anti-PSMA antibodies. A pathologist blinded to clinical and pathological data scored the immunoreactivity for these antibodies on a four-point scale (0 = no staining; 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining) and documented the distribution pattern. GRPR staining in lymph node metastases was seen in 85.7% of cases (18 of 21 cases), PSCA in 95.2% (20/21), and PSMA in 100% (21/21). GRPR in bone metastases was seen in 52.9% of cases (9/17), PSCA in 94.1% (16/17), and PSMA in 100% (17/17). We have shown for the first time that GRPR is expressed in the vast majority of lymph node metastases and in 52.9% of bone metastases of prostate cancer. PSCA and PSMA are both highly expressed in lymph node and bone metastases. Although PSCA and PSMA are mostly expressed in prostate cancer metastases, GRPR offers an interesting alternative target as it can be targeted relatively easy with peptide-based (radio)pharmaceuticals. (c) 2009 Wiley-Liss, Inc.

Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire

PubMed Central

Best, Katharine; Chain, Benny; Watkins, Chris

2015-01-01

The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the “danger” model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

Quantitative analysis of glycation and its impact on antigen binding

PubMed Central

Mo, Jingjie; Yan, Qingrong; Sokolowska, Izabela; Lewis, Michael J.; Hu, Ping

2018-01-01

ABSTRACT Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs. PMID:29436927

Epigenomic Promoter Alterations Amplify Gene Isoform and Immunogenic Diversity in Gastric Adenocarcinoma.

PubMed

Qamra, Aditi; Xing, Manjie; Padmanabhan, Nisha; Kwok, Jeffrey Jun Ting; Zhang, Shenli; Xu, Chang; Leong, Yan Shan; Lee Lim, Ai Ping; Tang, Qianqao; Ooi, Wen Fong; Suling Lin, Joyce; Nandi, Tannistha; Yao, Xiaosai; Ong, Xuewen; Lee, Minghui; Tay, Su Ting; Keng, Angie Tan Lay; Gondo Santoso, Erna; Ng, Cedric Chuan Young; Ng, Alvin; Jusakul, Apinya; Smoot, Duane; Ashktorab, Hassan; Rha, Sun Young; Yeoh, Khay Guan; Peng Yong, Wei; Chow, Pierce K H; Chan, Weng Hoong; Ong, Hock Soo; Soo, Khee Chee; Kim, Kyoung-Mee; Wong, Wai Keong; Rozen, Steven G; Teh, Bin Tean; Kappei, Dennis; Lee, Jeeyun; Connolly, John; Tan, Patrick

2017-06-01

Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms ( RASA3 ). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro , suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 therapeutic inhibition, and are associated with novel cancer-associated transcripts. By generating tumor-specific isoforms and decreasing tumor antigenicity, epigenomic promoter alterations may thus drive intrinsic tumorigenesis and also allow nascent cancers to evade host immunity. Significance: We apply epigenomic profiling to demarcate the promoter landscape of gastric cancer. Many tumor-specific promoters activate different promoters in the same gene, some generating pro-oncogenic isoforms. Tumor-specific promoters also reduce tumor antigenicity by causing relative depletion of immunogenic peptides, contributing to cancer immunoediting and allowing tumors to evade host immune attack. Cancer Discov; 7(6); 630-51. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 539 . ©2017 American Association for Cancer Research.

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

A paradigm for peptide vaccine delivery using viral epitopes encapsulated in degradable polymer hydrogel capsules.

PubMed

Chong, Siow-Feng; Sexton, Amy; De Rose, Robert; Kent, Stephen J; Zelikin, Alexander N; Caruso, Frank

2009-10-01

We report on the use of degradable polymer capsules as carriers for the delivery of oligopeptide antigens to professional antigen presenting cells (APCs). To achieve encapsulation, oligopeptide sequences were covalently linked to a negatively charged carrier polymer via biodegradable linkages and the resulting conjugate was then adsorbed onto amine-functionalized silica particles. These peptide-coated particles were then used as templates for the layer-by-layer (LbL) deposition of thiolated poly(methacrylic acid) (PMA(SH)) and poly(vinylpyrrolidone) (PVPON) multilayers. Removal of the silica core and disruption of the hydrogen bonding between PMA(SH) and PVPON by altering the solution pH yielded disulfide-stabilized PMA capsules that retain the encapsulated cargo in an oxidative environment. In the presence of a natural reducing agent, glutathione, cleavage of the disulfide bonds causes release of the peptide from the capsules. The developed strategy provides control over peptide loading into polymer capsules and yields colloidally stable micron- and submicron-sized carriers with uniform size and peptide loading. The conjugation and encapsulation procedures were proven to be non-degrading to the peptide vaccines. The peptide-loaded capsules were successfully used to deliver their cargo to APCs and activate CD8 T lymphocytes in a non-human primate model of SIV infection ex vivo. The reported approach represents a novel paradigm in the delivery of peptide vaccines and other therapeutic agents.

Peptide Probes Reveal a Hydrophobic Steric Ratchet in the Anthrax Toxin Protective Antigen Translocase.

PubMed

Colby, Jennifer M; Krantz, Bryan A

2015-11-06

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor and edema factor, into the host cytosol under the proton motive force. Protein translocation under a proton motive force is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between the lethal factor amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide-clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides, and while these steric interactions may make a peptide translocate poorly, in the context of folded domains, they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

PubMed

Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz

2013-01-25

An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Antigen processing in vivo and the elicitation of primary CTL responses.

PubMed

Restifo, N P; Bacík, I; Irvine, K R; Yewdell, J W; McCabe, B J; Anderson, R W; Eisenlohr, L C; Rosenberg, S A; Bennink, J R

1995-05-01

CD8+ T lymphocytes (TCD8+) play an important role in cellular immune responses. TCD8+ recognize MHC class I molecules complexed to peptides of 8 to 10 residues derived largely from cytosolic proteins. Proteins are generally thought to be fragmented in the cytoplasm and delivered to nascent class I molecules in the endoplasmic reticulum (ER) by a peptide transporter encoded by the MHC. To explore the extent to which TCD8+ induction in vivo is limited by proteolysis or peptide transport into the ER, mice were immunized with recombinant vaccinia viruses containing mini-genes encoding antigenic peptides (bypassing the need for proteolysis), or these peptides with a NH2-terminal ER insertion sequence (bypassing the requirements for both proteolysis and transport). Additionally, mice were immunized with recombinant vaccinia viruses encoding rapidly degraded fragments of proteins. We report that limitations in induction of TCD8+ responses vary among Ags: for some, full length proteins are as immunogenic as other forms tested; for others, maximal responses are induced by peptides or by peptides targeted to the ER. Most importantly, in every circumstance examined, targeting peptides to the ER never diminished, and in some cases greatly enhanced, the TCD8+ immune response and provide an important alternative strategy in the design of live viral or naked DNA vaccines for the treatment of cancer and infectious diseases.

A synthetic peptide vaccine directed against the 2ß2-2ß3 loop of domain 2 of protective antigen protects rabbits from inhalation anthrax.

PubMed

Oscherwitz, Jon; Yu, Fen; Cease, Kemp B

2010-09-15

The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.

An MSI Tumor Specific Frameshift Mutation in a Coding Microsatellite of MSH3 Encodes for HLA-A0201-Restricted CD8+ Cytotoxic T Cell Epitopes

PubMed Central

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Background Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI+ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. Methodology/Principal Findings FSP-specific CD8+ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI+ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. Conclusions We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were 386-FLLALWECSL (FSP18) and 387-LLALWECSL (FSP19) as well as 403-IVSRTLLLV (FSP23) and 402-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI+-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes. PMID:22110587

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells

PubMed Central

Kumar, Naveen; Biswas, Sunetra; Jumani, Rajiv S.; Jain, Chandni; Rani, Rajni; Aggarwal, Bharti; Singh, Jaya; Kotnur, Mohan Rao; Sridharan, Anand

2016-01-01

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4+ and CD8+ T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8+ T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8+ PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4+ T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8+ T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease. PMID:26843486

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

PubMed

Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

2012-07-01

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

[Experimental study of candidate vaccines against variable or quasi-species pathogenes: multiepitopic synthetic peptide antigenes and new receptor-guiding adjuvants].

PubMed

Ignat'eva, G A; Maksiutov, A Z; L'vov, V L; Kolobov, A A; Ignat'ev, T I

2011-01-01

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

PubMed Central

Tewalt, Eric F.; Grant, Jean M.; Granger, Erica L.; Palmer, Douglas C.; Heuss, Neal D.; Gregerson, Dale S.; Restifo, Nicholas P.; Norbury, Christopher C.

2009-01-01

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines. PMID:19478869

Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells.

PubMed

Carabali-Isajar, Mary Lilian; Ocampo, Marisol; Rodriguez, Deisy Carolina; Vanegas, Magnolia; Curtidor, Hernando; Patarroyo, Manuel Alfonso; Patarroyo, Manuel Elkin

2018-05-15

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 ( 155 VLAAYVYSLDNKRLWSNLDT 173 ) and 39266 ( 174 APSNETLVKTFSPGEQVTTY 192 ). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

PubMed

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

2017-01-01

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

Proteolysis, proteasomes and antigen presentation

NASA Technical Reports Server (NTRS)

Goldberg, A. L.; Rock, K. L.

1992-01-01

Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.

Endogenous antigen processing drives the primary CD4+ T cell response to influenza

PubMed Central

Miller, Michael A.; Ganesan, Asha Purnima V.; Luckashenak, Nancy; Mendonca, Mark; Eisenlohr, Laurence C.

2015-01-01

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with MHC class II molecules. Alternative pathways of epitope production have been identified but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome, and gamma-interferon inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses. PMID:26413780

Encapsulating Immunostimulatory CpG Oligonucleotides in Listeriolysin O-Liposomes Promotes a Th1-Type Response and CTL Activity

PubMed Central

Andrews, Chasity D.; Huh, Myung-Sook; Patton, Kathryn; Higgins, Debbie; Van Nest, Gary; Ott, Gary; Lee, Kyung-Dall

2013-01-01

Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that co-encapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the co-encapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with either OVA-containing LLO-liposomes or OVA-ISS conjugates. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type. PMID:22376145

Fiber optic immunosensor for cross-linked fibrin concentration

NASA Astrophysics Data System (ADS)

Moskowitz, Samuel E.

2000-08-01

Working with calcium ions in the blood, platelets produce thromboplastin which transforms prothrombin into thrombin. Removing peptides, thrombin changes fibrinogen into fibrin. Cross-linked insoluble fibrin polymers are solubilized by enzyme plasmin found in blood plasma. Resulting D-dimers are elevated in patients with intravascular coagulation, deep venous thrombosis, pulmonary embolism, myocardial infarction, multiple trauma, cancer, impaired renal and liver functions, and sepsis. Consisting principally of a NIR 780 nm GaAlAs laser diode and a 800 nm avalanche photodiode (APD), the fiber-optic immunosensor can determined D-dimer concentration to levels <0.1 ng/ml. A capture monoclonal antibody to the antigen soluble cross-linked fibrin is employed. Immobilized at the tip of an optical fiber by avidin-biotin, the captured antigen is detected by a second antibody which is labeled with NN 382 fluorescent dye. An evanescent wave traveling on an excitation optical fiber excites the antibody-antigen fluorophore complex. Concentration of cross-linked fibrin is directly proportional to the APD measured intensity of fluorescence. NIR fluorescence has advantages of low background interference, short fluorescence lifetime, and large difference between excitation and emission peaks. Competitive ELISA test for D-dimer concentration requires trained personnel performing a time consuming operation.

Major antigenic determinants of F and ColB2 pili.

PubMed Central

Finlay, B B; Frost, L S; Paranchych, W; Parker, J M; Hodges, R S

1985-01-01

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule. PMID:2409073

Discrimination Between Human Leukocyte Antigen Class I-Bound and Co-Purified HIV-Derived Peptides in Immunopeptidomics Workflows

PubMed Central

Partridge, Thomas; Nicastri, Annalisa; Kliszczak, Anna E.; Yindom, Louis-Marie; Kessler, Benedikt M.; Ternette, Nicola; Borrow, Persephone

2018-01-01

Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of “irrelevant” membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated into candidate vaccines. These results have wider implications for HLA epitope discovery from abundant or membrane-associated antigens by immunopeptidomics in the context of infectious diseases, cancer, and autoimmunity. PMID:29780384

Hepatitis C Virus Antigenic Convergence

PubMed Central

Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury

2012-01-01

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779

Supramolecular peptide hydrogel adjuvanted subunit vaccine elicits protective antibody responses against West Nile virus.

PubMed

Friedrich, Brian M; Beasley, David W C; Rudra, Jai S

2016-11-04

A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine.

PubMed

Tipu, Hamid Nawaz

2016-02-01

To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.

[Planar molecular arrangements aid the design of MHC class II binding peptides].

PubMed

Cortés, A; Coral, J; McLachlan, C; Benítez, R; Pinilla, L

2017-01-01

The coupling between peptides and MHC-II proteins in the human immune system is not well understood. This work presents an evidence-based hypothesis of a guiding intermolecular force present in every human MHC-II protein (HLA-II). Previously, we examined the spatial positions of the fully conserved residues in all HLA-II protein types. In each one, constant planar patterns were revealed. These molecular planes comprise of amino acid groups of the same chemical species (for example, Gly) distributed across the protein structure. Each amino acid plane has a unique direction and this directional element offers spatial selectivity. Constant within all planes, too, is the presence of an aromatic residue possessing electrons in movement, leading the authors to consider that the planes generate electromagnetic fields that could serve as an attractive force in a single direction. Selection and attraction between HLA-II molecules and antigen peptides would, therefore, be non-random, resulting in a coupling mechanism as effective and rapid as is clearly required in the immune response. On the basis of planar projections onto the HLA-II groove, modifications were made by substituting the key residues in the class II-associated invariant chain peptide-a peptide with a universal binding affinity-resulting in eight different modified peptides with affinities greater than that of the unmodified peptide. Accurate and reliable prediction of MHC class II-binding peptides may facilitate the design of universal vaccine-peptides with greatly enhanced binding affinities. The proposed mechanisms of selection, attraction and coupling between HLA-II and antigen peptides are explained further in the paper.

Masked Chimeric Antigen Receptor for Tumor-Specific Activation.

PubMed

Han, Xiaolu; Bryson, Paul D; Zhao, Yifan; Cinay, Gunce E; Li, Si; Guo, Yunfei; Siriwon, Natnaree; Wang, Pin

2017-01-04

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. In vitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy in vivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

PubMed

Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

2015-08-25

Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

PubMed

Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

2013-09-13

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, F; Rasaee, M J; Forouzandeh Moghadam, M; Allameh, A A; Sadroddiny, E

2004-06-01

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

PubMed

Emmanouil, Mary; Tseveleki, Vivian; Triantafyllakou, Iro; Nteli, Agathi; Tselios, Theodore; Probert, Lesley

2018-01-31

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP 87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP 72-85 -induced EAE in Lewis rats. The Lys 91 and Pro 96 of MBP 87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala 96 ]MBP 87-99 , cyclo(87-99)[Ala 91,96 ]MBP 87-99 and cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 , but not wild-type linear MBP 87-99 , strongly inhibited MBP 72-85 -induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II

PubMed Central

Rehm, Kristina E; Connor, Ramsey F; Jones, Gwendolyn J B; Yimbu, Kenneth; Mannie, Mark D; Roper, Rachel L

2009-01-01

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-γ, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC. PMID:20067538

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

PubMed Central

Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

2012-01-01

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

Generation of human anti-MUC3 IgG antibodies after in vitro immunization of naive peripheral blood B-lymphocytes.

PubMed

Baritaki, S; Zafiropoulos, A; Georgopoulos, E; Souris, S; Krambovitis, E

2001-04-01

It has been demonstrated that IgG antibodies can be generated to self-antigen peptides as well as against viral antigens by an antigen-specific in vitro immunization system of resting human peripheral B-lymphocytes. Using a synthetic peptide from the consensus variable tandem-repeat region of the MUC3 mucin (TSSITTTGTTSHSTPSP) as the B cell epitope, we immunized blood donor B-lymphocytes in vitro and tested for MUC3-specific antibodies by ELISA. After the primary activation step all antibodies were IgM. At the end of the secondary immunization step we obtained 1.8% (21/1138) of the cultures with IgG-switched antibodies. In a competitive inhibition ELISA using the MUC1, MUC2, MUC3, MUC4 and PIP2 peptides, only one culture (F8.1) gave satisfactory specific inhibition. Using this antibody in fluorometric studies, it stained cells from two colon carcinoma cell lines predominantly in the cytoplasm, whereas those from a breast cancer cell line stained predominantly the cell surface. In a preliminary immunohistological evaluation with formalin-fixed sections, the antibody appeared to moderately stain colon sections, but not breast sections or lymph node. This method of in vitro immunization may be a useful tool in generating IgG antibodies specific to self-antigens and could find applications in tumour targeting and immunotherapy.

Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

PubMed

Al-Attiyah, R; Mustafa, A S

2004-01-01

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

PubMed

Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A

2015-03-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mapping Antigenic Motifs in the Trypomastigote Small Surface Antigen from Trypanosoma cruzi

PubMed Central

Balouz, Virginia; Cámara, María de los Milagros; Cánepa, Gaspar E.; Carmona, Santiago J.; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán

2015-01-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. PMID:25589551

Peptide/protein vaccine delivery system based on PLGA particles.

PubMed

Allahyari, Mojgan; Mohit, Elham

2016-03-03

Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted.

Peptide/protein vaccine delivery system based on PLGA particles

PubMed Central

Allahyari, Mojgan; Mohit, Elham

2016-01-01

abstract Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

Recent advances in Major Histocompatibility Complex (MHC) class I antigen presentation: Plastic MHC molecules and TAPBPR-mediated quality control

PubMed Central

van Hateren, Andy; Bailey, Alistair; Elliott, Tim

2017-01-01

We have known since the late 1980s that the function of classical major histocompatibility complex (MHC) class I molecules is to bind peptides and display them at the cell surface to cytotoxic T cells. Recognition by these sentinels of the immune system can lead to the destruction of the presenting cell, thus protecting the host from pathogens and cancer. Classical MHC class I molecules (MHC I hereafter) are co-dominantly expressed, polygenic, and exceptionally polymorphic and have significant sequence diversity. Thus, in most species, there are many different MHC I allotypes expressed, each with different peptide-binding specificity, which can have a dramatic effect on disease outcome. Although MHC allotypes vary in their primary sequence, they share common tertiary and quaternary structures. Here, we review the evidence that, despite this commonality, polymorphic amino acid differences between allotypes alter the ability of MHC I molecules to change shape (that is, their conformational plasticity). We discuss how the peptide loading co-factor tapasin might modify this plasticity to augment peptide loading. Lastly, we consider recent findings concerning the functions of the non-classical MHC I molecule HLA-E as well as the tapasin-related protein TAPBPR (transporter associated with antigen presentation binding protein-related), which has been shown to act as a second quality-control stage in MHC I antigen presentation. PMID:28299193

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

PubMed

Omidfar, K; Rasaee, M J; Modjtahedi, H; Forouzandeh, M; Taghikhani, M; Golmakani, N

2004-01-01

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications. Copyright 2004 S. Karger AG, Basel.

Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

PubMed

Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua

2015-08-01

T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

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

PubMed

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

2007-07-01

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

A peptide-major histocompatibility complex II chimera favors survival of pancreatic beta-islets grafted in type 1 diabetic mice.

PubMed

Casares, Sofia; Lin, Marvin; Zhang, Nan; Teijaro, John R; Stoica, Cristina; McEvoy, Robert; Farber, Donna L; Bona, Constantin; Brumeanu, Teodor D

2008-06-27

Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.

[Development of Peptide Vaccines for Triple-Negative Breast Cancer Treatment].

PubMed

Toh, Uhi; Saku, Shuko; Okabe, Mina; Iwakuma, Nobutaka; Kimitsuki, Yuko; Akashi, Momoko; Ogo, Etsuyo; Yamada, Akira; Shichijo, Shigeki; Itoh, Kyogo; Akagi, Yoshito

2016-10-01

Our previous phase II clinical trial showed that therapeutically selected personalized peptide vaccines(PPVs)were effective at boosting anticancer immunity; the immune response after PPV was associated with a clinical outcome as a prognostic factor for metastatic breast cancer(mBC). We conducted an early phase II study to evaluate the safety and efficacy of a new regimen using multiple peptide vaccines(KRM-19)for patients with metastatictriple -negative breast cancer. KRM-19 consisted of 19 mixed peptides chosen from the previously reported 31 PPVs according to their anti-tumor immunologiceffec ts and safety profiles for patients with mBC. All patients had histologically confirmed measurable ER-PgR-HER2- mBC and their human leukocyte antigen(HLA) / -A molecules were A2, A3, A11, A24, A26, A31, or A33. KRM-19(19mg/mL)was administrated subcutaneously every week for a total of 6 doses. Concurrent conventional chemo- and/or endocrine therapy were not permitted during treatment. This was an open-label, early phase II study. The primary endpoint was safety and anti-tumor immunologic effect, while the secondary endpoints were clinical responses and progression-free survival(PFS). The estimated enrollment was 10-15 and 8 patients were enrolled(Clinical trial registry number: UMIN000014616). Measurement of peptide-specific cytotoxic T lymphocyte and IgG responses were conducted before and after vaccination. The correlation between PFS and the increased IgG response and/or CTL levels were investigated.

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

PubMed

Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

2009-05-15

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

76 FR 66726 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-27

...-A12 peptide antigens. The TCRs recognize these antigens in the context of major histocompatibility... proteins to mediate tumor growth and spreading. The MAGE-A proteins are some of the most widely expressed... adapted to the depletion of hormones and continue to grow. Abnormal androgen receptor signaling is known...

Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization

PubMed Central

Brune, Karl D.; Leneghan, Darren B.; Brian, Iona J.; Ishizuka, Andrew S.; Bachmann, Martin F.; Draper, Simon J.; Biswas, Sumi; Howarth, Mark

2016-01-01

Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices. PMID:26781591

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

PubMed Central

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

2012-01-01

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

Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.

PubMed

Okumura, Hideo; Noguchi, Yuji; Uenaka, Akiko; Aji, Toshiki; Ono, Toshiro; Nakagawa, Kazuhiko; Aoe, Motoi; Shimizu, Nobuyoshi; Nakayama, Eiichi

2005-01-01

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.

Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice.

PubMed

Iwama, Tatsuaki; Uchida, Tetsuya; Sawada, Yu; Tsuchiya, Nobuhiro; Sugai, Shiori; Fujinami, Norihiro; Shimomura, Manami; Yoshikawa, Toshiaki; Zhang, Rong; Uemura, Yasushi; Nakatsura, Tetsuya

2016-01-01

Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Humoral immune profiling of mycobacterial antigen recognition in sarcoidosis and Löfgren's syndrome using high-content peptide microarrays.

PubMed

Ferrara, Giovanni; Valentini, Davide; Rao, Martin; Wahlström, Jan; Grunewald, Johan; Larsson, Lars-Olof; Brighenti, Susanna; Dodoo, Ernest; Zumla, Alimuddin; Maeurer, Markus

2017-03-01

Sarcoidosis is considered an idiopathic granulomatous disease, although similar immunological and clinical features with tuberculosis (TB) suggest mycobacterial involvement in its pathogenesis. High-content peptide microarrays (HCPM) may help to decipher mycobacteria-specific antibody reactivity in sarcoidosis. Serum samples from patients with sarcoidosis, Löfgren's syndrome, and TB, as well as from healthy individuals (12/group), were tested on HCPM containing 5964 individual peptides spanning 154 Mycobacterium tuberculosis proteins displayed as 15-amino acid stretches. Inclusion/exclusion and significance analyses were performed according to published methods. Each study group recognized 68-78% M. tuberculosis peptides at least once. M. tuberculosis epitope recognition by sarcoidosis patient sera was 42.7%, and by TB patient sera was 39.1%. Seven and 16 peptides were recognized in 9/12 (75%) and 8/12 (67%) sarcoidosis patient sera but not in TB patient sera, respectively. Nine (75%) and eight (67%) out of twelve TB patient sera, respectively recognized M. tuberculosis peptides that were not recognized in sarcoidosis patient sera. Specific IgG recognition patterns for M. tuberculosis antigens in sarcoidosis patients re-affirm mycobacterial involvement in sarcoidosis, providing biologically relevant targets for future studies pertaining to diagnostics and immunotherapy. Copyright © 2017. Published by Elsevier Ltd.

Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201 for dendritic cell based immunotherapy of gliomas.

PubMed

Wu, An-hua; Xiao, Jing; Anker, Lars; Hall, Walter A; Gregerson, Dale S; Cavenee, Webster K; Chen, Wei; Low, Walter C

2006-01-01

The type III variant of the epidermal growth factor receptor (EGFRvIII) mutation is present in 20-25% of patients with glioblastoma multiforme (GBM). EGFRvIII is not expressed in normal tissue and is therefore a suitable candidate antigen for dendritic cell (DC) based immunotherapy of GBM. To identify the antigenic epitope(s) that may serve as targets for EGFRvIII-specific cytotoxic T lymphocytes (CTLs), the peptide sequence of EGFRvIII was screened with two software programs to predict candidate epitopes restricted by the major histocompatibility complex class I subtype HLA-A0201, which is the predominant subtype in most ethnic groups. Three predicted peptides were constructed and loaded to mature human DCs generated from peripheral blood monocytes. Autologous CD8+ T cells were stimulated in vitro with the EGFRvIII peptide-pulsed DCs. One of the three peptides was found to induce EGFRvIII-specific CTLs as demonstrated by IFN-gamma production and cytotoxicity against HLA-A0201+ EGFRvIII transfected U87 glioma cells. These results suggest that vaccination with EGFRvIII peptide-pulsed DCs or adoptive transfer of in vitro elicited EGFRvIII-specific CTLs by EGFRvIII peptide-pulsed DCs are potential approaches to the treatment of glioma patients.

Dynamics of major histocompatibility complex class I association with the human peptide-loading complex.

PubMed

Panter, Michaela S; Jain, Ankur; Leonhardt, Ralf M; Ha, Taekjip; Cresswell, Peter

2012-09-07

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.

Immunomagnetic separation of human myeloperoxidase using an antibody-mimicking peptide identified by phage display.

PubMed

Yun, Soi; Ryu, Hyunmin; Lee, E K

2017-09-10

Phage display biopanning is a powerful in vitro selection process for screening and identifying peptides that bind to a target protein of interest. With the aim of replacing antibodies in immuno-diagnostic applications, we identified peptides whose binding characteristics mimicked those of anti-human myeloperoxidase (hMPO), a biomarker for acute cardiac diseases. Based on ELISA results from four phage clones, we selected and chemically synthesized a 12-mer peptide (SYIEPPERHRHR). Quartz crystal microbalance and surface plasmon resonance analyses revealed that the molar binding equilibrium ratio of the synthesized peptide was 0.023, approximately 43-fold lower than that of the anti-hMPO antibody. The dissociation constant (K d ) was 57nM, which was comparable to that of the native antibody (83nM). Next, we biotinylated the peptide at its N-terminus and attached the biotinylated peptide to the surface of streptavidin-coated magnetic particles to assess its ability to selectively capture hMPO. The binding equilibrium data were similar to the previous analyses; specifically, around 0.021mol peptide bound to 1mol of hMPO. Antigen capture was found to be selective and to be relatively little influenced by the presence of human serum albumin (HSA), an abundant constituent of serum. Our work demonstrates the potential of immunomagnetic isolation to achieve selective capture of a low-concentration antigen from complex solutions such as serum. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibody response of Schistosoma mansoni-infected human subjects to the recombinant P28 glutathione-S-transferase and to synthetic peptides.

PubMed

Auriault, C; Gras-Masse, H; Pierce, R J; Butterworth, A E; Wolowczuk, I; Capron, M; Ouma, J H; Balloul, J M; Khalife, J; Neyrinck, J L

1990-09-01

The 28-kilodalton antigen of Schistosoma mansoni has been previously described as having importance as the basis for a potential vaccine. The P28 recombinant molecule and three peptides derived from its primary sequence, namely the 24-43, 115-131, and 140-153 peptides, have been tested to evaluate the humoral responses of Kenyan school children previously classified as susceptible or resistant to reinfection after chemotherapy. We report here that the P28 molecule and two of the peptides studied (peptides 115-131 and 140-153) can be used for detecting specific immunoglobulin G (IgG), IgE, and IgA antibodies. Moreover, the IgG4 response of the susceptible population was significantly greater than that of the resistant group, whereas no differences between the two populations were noticed in total IgG anti-P28 antibodies. This suggested that IgG4 could play a role in the lack of immunity of susceptible patients. A strong IgG3 response restricted to the 140-153 peptide was observed but did not discriminate between the resistant and susceptible populations. In contrast, a marked increase in the IgA response to the 140-153 peptide epitope(s) in sera of the resistant population was noticed. Taken together, these results suggest that the P28 antigen and two of the three peptides selected could give predictive information about the development of the disease or the efficiency of vaccination with P28 as the immunogen.

Peptides in melanoma therapy.

PubMed

Mocellin, Simone

2012-01-01

Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

High Throughput T Epitope Mapping and Vaccine Development

PubMed Central

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

2010-01-01

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

Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics.

PubMed

Smith, Shanna J; Hickey, Robert J; Malkas, Linda H

2016-01-01

Human DNA replication and repair is a highly coordinated process involving the specifically timed actions of numerous proteins and enzymes. Many of these proteins require interaction with proliferating cell nuclear antigen (PCNA) for activation within the process. The interdomain connector loop (IDCL) of PCNA provides a docking site for many of those proteins, suggesting that this region is critically important in the regulation of cellular function. Previous work in this laboratory has demonstrated that a peptide mimicking a specific region of the IDCL (caPeptide) has the ability to disrupt key protein-protein interactions between PCNA and its binding partners, thereby inhibiting DNA replication within the cells. In this study, we confirm the ability of the caPeptide to disrupt DNA replication function using both intact cell and in vitro DNA replication assays. Further, we were able to demonstrate that treatment with caPeptide results in a decrease of polymerase δ activity that correlates with the observed decrease in DNA replication. We have also successfully developed a surface plasmon resonance (SPR) assay to validate the disruption of the PCNA-pol δ interaction with caPeptide.

ABC transporters and immunity: mechanism of self-defense.

PubMed

Hinz, Andreas; Tampé, Robert

2012-06-26

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.

Grafting of a peptide probe for Prostate-Specific Antigen detection using diazonium electroreduction and click chemistry.

PubMed

Strzemińska, I; Sainte Rose Fanchine, S; Anquetin, G; Reisberg, S; Noël, V; Pham, M C; Piro, B

2016-07-15

The main objective of this work was to validate a label-free electrochemical method of protein detection using peptides as capture probes. As a proof-of-concept, we used a 7 amino acids sequence (HSSKLQL) specific for Prostate Specific Antigen. We investigated various electrografting conditions of two anilines (2-[(4-aminophenyl)sulfanyl]-8-hydroxy-1,4-naphthoquinone and 4-azidoaniline) further converted in situ into their corresponding diazonium salts on glassy carbon electrodes. It was demonstrated that the best method to obtain a mixed layer is the simultaneous electroreduction of the two diazonium salts. 4-azidoaniline was used to covalently immobilize the ethynyl-functionalized peptide probe by click coupling, and the hydroxynaphthoquinone derivative plays the role of electrochemical transducer of the peptide-protein recognition. The proteolytic activity of PSA towards a small peptide substrate carrying streptavidin at its distal end was also investigated to design an original sensing architecture leading to a reagentless, label free, and "signal-on" PSA sensor. Without optimization, the limit of quantification can be estimated in the nM to pM range. Copyright © 2016 Elsevier B.V. All rights reserved.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis.

PubMed

Meshgi, Behnam; Jalousian, Fatemeh; Fathi, Saeid; Jahani, Zahra

2018-06-01

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 μg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites. Copyright © 2018 Elsevier Inc. All rights reserved.

Unusual antigen presentation offers new insight into HIV vaccine design.

PubMed

McMichael, Andrew J; Picker, Louis J

2017-06-01

Recent findings with a rhesus monkey cytomegalovirus based simian immunodeficiency virus vaccine have identified strong CD8+ T cell responses that are restricted by MHC-E. Also mycobacteria specific CD8+ T cells, that are MHC-E restricted, have been identified. MHC-E therefore can present a wide range of epitope peptides to CD8+ T cells, alongside its well defined role in presenting a conserved MHC-class I signal peptide to the NKG2A/C-CD94 receptor on natural killer cells. Here we explore the antigen processing pathways involved in these atypical T cell responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vaccine Adjuvant Incorporation Strategy Dictates Peptide Amphiphile Micelle Immunostimulatory Capacity.

PubMed

Zhang, Rui; Kramer, Jake S; Smith, Josiah D; Allen, Brittany N; Leeper, Caitlin N; Li, Xiaolei; Morton, Logan D; Gallazzi, Fabio; Ulery, Bret D

2018-06-01

Current vaccine research has shifted from traditional vaccines (i.e., whole-killed or live-attenuated) to subunit vaccines (i.e., protein, peptide, or DNA) as the latter is much safer due to delivering only the bioactive components necessary to produce a desirable immune response. Unfortunately, subunit vaccines are very weak immunogens requiring delivery vehicles and the addition of immunostimulatory molecules termed adjuvants to convey protective immunity. An interesting type of delivery vehicle is peptide amphiphile micelles (PAMs), unique biomaterials where the vaccine is part of the nanomaterial itself. Due to the modularity of PAMs, they can be readily modified to deliver both vaccine antigens and adjuvants within a singular construct. Through the co-delivery of a model antigenic epitope (Ovalbumin 319-340 -OVA BT ) and a known molecular adjuvant (e.g., 2,3-dipalmitoyl-S-glyceryl cysteine-Pam 2 C), greater insight into the mechanisms by which PAMs can exert immunostimulatory effects was gained. It was found that specific combinations of antigen and adjuvant can significantly alter vaccine immunogenicity both in vitro and in vivo. These results inform fundamental design rules that can be leveraged to fabricate optimal PAM-based vaccine formulations for future disease-specific applications. Graphical Abstract.

Peptide vaccination of patients with metastatic melanoma: improved clinical outcome in patients demonstrating effective immunization.

PubMed

Markovic, Svetomir N; Suman, Vera J; Ingle, James N; Kaur, Judith S; Pitot, Henry C; Loprinzi, Charles L; Rao, Ravi D; Creagan, Edward T; Pittelkow, Mark R; Allred, Jakob B; Nevala, Wendy K; Celis, Esteban

2006-08-01

Therapeutic peptide vaccines for melanoma continue to only demonstrate anecdotal success. We set out to evaluate the impact of low-dose GM-CSF emulsified in Montanide ISA-51 on the immunogenicity of HLA-A2 restricted melanoma differentiation antigen peptide vaccines (MART-1, gp100 and tyrosinase) administered in separate subcutaneous injections. We conducted a randomized phase II clinical trial of HLA-A2+ patients with metastatic melanoma that were immunized every 3 weeks with one of the following vaccine preparations: (A) peptides + Montanide ISA-51; (B) peptides + Montanide ISA-51 + GM-CSF (10 microg); (C) peptides + Montanide ISA-51 + GM-CSF (50 microg). Immunization efficacy was determined by quantification of vaccine specific tetramer positive cytotoxic T cells in peripheral blood. Global assessment of immune competence was ascertained using DTH testing to common recall antigens as well as peripheral blood immunophenotyping. Twenty-five eligible patients were equally distributed across all 3 treatment groups. Only 9 patients demonstrated evidence of immunization. Most commonly, immune response was achieved to the gp100 peptide. The addition of low-dose GM-CSF did not impact immunization efficacy. DTH reactivity to Candida appeared predictive of successful immunization. Successful immunization with the peptide vaccines was associated with improved clinical outcomes. The addition of low dose GM-CSF to peptide vaccines did not enhance immunogenicity. Higher doses of GM-CSF may be needed to achieve this effect and this is a testable hypothesis. Likewise, better patient selection based on immunologic status (DTH reactivity) may be helpful to better understand the clinical impact of therapeutic cancer vaccines.

Antigen-dependent fluorescence response of anti-c-Myc Quenchbody studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Mori, Yoshiharu; Okumura, Hisashi; Watanabe, Takayoshi; Hohsaka, Takahiro

2018-04-01

We performed metadynamics molecular dynamics simulations to reveal mechanism of antigen-dependent fluorescence response observed for site-specifically fluorescent-labeled single-chain antibody against c-Myc peptide antigen. We found that VH and VL bind with each other only when the antigen exists and that the fluorophore labeled at the N-terminus of VH interacts with Trp103 most stably. These results support the mechanism proposed from previous experiments: In the absence of antigen, Trp residues are partially exposed at the interface of VH and quench the fluorophore. In the presence of antigen, the Trp residues are buried between VH and VL , and the quenching is eliminated.

Preparation and evaluation of the tumor-specific antigen-derived synthetic mucin 1 peptide: A potential candidate for the targeting of breast carcinoma.

PubMed

Okarvi, Subhani M; Al Jammaz, Ibrahim

2016-07-01

The goal of this study was to prepare a synthetic peptide derived from breast tumor associated antigen and to evaluate its potential as a breast cancer imaging agent. A mucin 1 derived peptide was synthesized by solid-phase peptide synthesis and examined for its radiochemical and metabolic stability. The tumor cell binding affinity of (99m)Tc-MUC1 peptide was investigated on MUC1-positive T47D and MCF7 breast cancer cell lines. In vivo biodistribution was studied in normal Balb/c mice and in vivo tumor targeting and imaging in MCF7 and T47D tumor-bearing nude mice. The synthesized MUC1-derived peptide displayed high radiochemical and metabolic stability. In vitro tumor cell-binding on T47D and MCF7 cell lines demonstrated high affinity of (99m)Tc-MUC1 peptide towards human breast cancer cells (binding affinities in nanomolar range). Pharmacokinetic studies performed on Balb/c mice are characterized by an efficient clearance from the blood and excretion predominantly through the urinary system. In vivo tumor uptake in nude mice with MCF7 tumor xenografts was 2.77±0.63% ID/g as early as 1h p.i. whereas in nude mice with T47D human ductal breast epithelial cancer cells, the accumulation in the tumor was found to be 2.65±0.54% ID/g at 1h p.i. Also tumor lesion was detectable in γ-camera imaging. The tumor uptake values were always higher than the blood and muscle uptake, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. A low to moderate (<5% ID/g) accumulation and retention of (99m)Tc-MUC1 was found in the major organs (i.e., lungs, stomach, liver, intestines, kidneys, etc.) in both normal and tumor-bearing mice. This study suggests that (99m)Tc-MUC1 tumor-antigen peptide may be a potential candidate for the targeted imaging of MUC1-positive human tumors and warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

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

T-cells from HLA-B*57:01+ human subjects are activated with abacavir through two independent pathways and induce cell death by multiple mechanisms.

PubMed

Bell, Catherine C; Faulkner, Lee; Martinsson, Klara; Farrell, John; Alfirevic, Ana; Tugwood, Jonathan; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

2013-05-20

Susceptibility to abacavir hypersensitivity has been attributed to possession of the specific human leukocyte antigen allele HLA-B*57:01. HLA-B*57:01-restricted activation of CD8+ T-cells provides a link between the genetic association and the iatrogenic disease. The objectives of this study were to characterize the functionality of drug-responsive CD8+ T-cell clones generated from HLA-B*57:01+ drug-naive subjects and to explore the relationship between abacavir accumulation in antigen presenting cells and the T-cell response. Seventy-four CD8+ clones expressing different Vβ receptors were shown to proliferate and kill target cells via different mechanisms when exposed to abacavir. Certain clones were activated with abacavir in the absence of antigen presenting cells. Analysis of the remaining clones revealed two pathways of drug-dependent T-cell activation. Overnight incubation of antigen presenting cells with abacavir, followed by repeated washing to remove soluble drug, activated approximately 50% of the clones, and the response was blocked by glutaraldehyde fixation. In contrast, a 1 h antigen presenting cell pulse did not activate any of the clones. Accumulation of abacavir in antigen presenting cells was rapid (less than 1 h), and the intracellular concentrations were maintained for 16 h. However, intracellular abacavir was not detectable by mass spectrometry after pulsing. These data suggest that T-cells can be activated by abacavir through a direct interaction with surface and intracellular major histocompatibility complex (MHC) molecules. With the former, abacavir seemingly participates in the MHC T-cell receptor binding interaction. In contrast, the latter pathway likely involves MHC binding peptides displayed as a consequence of abacavir exposure, but not abacavir itself.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal.

PubMed

Bobosha, Kidist; Tang, Sheila Tuyet; van der Ploeg-van Schip, Jolien J; Bekele, Yonas; Martins, Marcia V S B; Lund, Ole; Franken, Kees L M C; Khadge, Saraswoti; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Hussien, Jemal; Thapa, Pratibha; Kunwar, Chhatra B; Hagge, Deanna A; Aseffa, Abraham; Pessolani, Maria Cristina Vidal; Pereira, Geraldo M B; Ottenhoff, Tom H M; Geluk, Annemieke

2012-12-01

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Lewis Y Antigen as a Target for Breast Cancer Therapy

DTIC Science & Technology

1996-09-01

have shown that a synthetic peptide can mimic the capsular polysaccharide of N. meningitis serogroup C (MCP) in that it induces an anti-MCP immune...intervening residue. All these sequences resemble the peptide we have identified as a mimic of the group C meningococcal polysaccharide . The immunological...Group C Polysaccharide ct(2-9)sialic acid The sequence similarities among the putative motifs suggest that antibodies raised to this peptide set might

Structural and dynamic features of HLA-B27 subtypes.

PubMed

Uchanska-Ziegler, Barbara; Ziegler, Andreas; Schmieder, Peter

2013-07-01

The differential association of HLA-B27 subtypes with ankylosing spondylitis provides the rationale for a comparative investigation of these proteins. Results from the last 2 years of research on minimally distinct HLA-B27 subtypes, primarily using biochemical and biophysical techniques, are presented and discussed. We summarize evidence that micropolymorphisms within the molecules' peptide-binding groove influence wide-ranging biochemical, biophysical and antigenic properties of HLA-B27 molecules, and suggest that distinct, subtype and peptide-dependent dynamics of peptide - heavy chain - β(2)-microglobulin heterotrimers could be instrumental for an understanding of the initiation of disease processes that are connected with certain HLA-B27 subtypes. The results indicate that mAbs that bind only to structurally distinguishable subsets of HLA-B27 molecules as well as techniques that assess the flexibility of these antigens may hold the key to comprehend molecular events contributing to the initial stages of disease pathogenesis in spondyloarthropathies.

Quantitating protein synthesis, degradation, and endogenous antigen processing.

PubMed

Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

2003-03-01

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.

Glioma antigen.

PubMed

Toda, Masahiro

2012-01-01

Because several antigenic peptides of human tumors that are recognized by T-lymphocytes have been identified, immune responses against cancer can now be artificially manipulated. Furthermore, since T-lymphocytes have been found to play an important role in the rejection of tumors by the host and also to have antigen-specific proliferative potentials and memory mechanisms, T-lymphocytes are thought to play a central role in cancer vaccination. Although multidisciplinary therapies have been attempted for the treatment of gliomas, the results remain unsatisfactory. For the development of new therapies against gliomas, it is required to identify tumor antigens as targets for specific immunotherapy. In this chapter, recent progress in research on glioma antigens is described.

Distinct Inflammatory Profiles of Myelin-Reactive T cells from Patients with Multiple Sclerosis

PubMed Central

Cao, Yonghao; Goods, Brittany A.; Raddassi, Khadir; Nepom, Gerald T.; Kwok, William W.; Love, J. Christopher; Hafler, David A.

2015-01-01

Myelin-reactive T cells have been identified in patients with multiple sclerosis (MS) and healthy subjects with comparable frequencies, but the functional programs of self-reactive T cells that promote disease remain unknown. A total of 13,324 T cell libraries generated from blood of 23 patients and 22 healthy controls were interrogated for reactivity to myelin antigens. Libraries derived from CCR6+ myelin-reactive T cells from patients with MS exhibited significantly enhanced production of IFN-γ, IL-17, and GM-CSF compared to healthy controls. Single-cell clones isolated by MHC/peptide tetramers from CCR6+ T cell libraries also secreted more pro-inflammatory cytokines while clones isolated from controls secreted more IL-10. The transcriptomes of myelin-specific CCR6+ T cells from patients with MS were distinct from those derived from healthy controls, and of note, were enriched in Th17-induced experimental autoimmune encephalitis (EAE) gene signatures and gene signatures derived from Th17 cells isolated other human autoimmune diseases. These data, although not casual, imply that functional differences between antigen specific T cells from MS and healthy controls is fundamental to disease development and support the notion that IL-10 production from myelin-reactive T cells may act to limit disease progression, or even pathogenesis. PMID:25972006

Mass Spectrometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein*

PubMed Central

de Laurentiis, Annamaria; Gaspari, Marco; Palmieri, Camillo; Falcone, Cristina; Iaccino, Enrico; Fiume, Giuseppe; Massa, Ornella; Masullo, Mariorosario; Tuccillo, Franca Maria; Roveda, Laura; Prati, Ubaldo; Fierro, Olga; Cozzolino, Immacolata; Troncone, Giancarlo; Tassone, Pierfrancesco; Scala, Giuseppe; Quinto, Ileana

2011-01-01

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis. PMID:21372249

A Novel Method Linking Antigen Presentation by Human Monocyte-Derived Macrophages to CD8+ T Cell Polyfunctionality

PubMed Central

Short, Kirsty R.; Grant, Emma J.; Vissers, Marloes; Reading, Patrick C.; Diavatopoulos, Dimitri A.; Kedzierska, Katherine

2013-01-01

To understand the interactions between innate and adaptive immunity, and specifically how virally infected macrophages impact T cell function, novel assays examining the ability of macrophages to present antigen to CD8+ T cells are needed. In the present study, we have developed a robust in vitro assay to measure how antigen presentation by human monocyte-derived macrophages (MDMs) affects the functional capacity of autologous CD8+ T cells. The assay is based on the polyfunctional characteristics of antigen-specific CD8+ T cells, and is thus called a Mac-CD8 Polyfunctionality Assay. Following purification of monocytes and their maturation to MDMs, MDMs were pulsed with an antigenic peptide to be presented to CD8+ T cells. Peptide-pulsed MDMs were then incubated with antigen-specific CD8+ T cells in order to assess the efficacy of antigen presentation to T cells. CD8+ T cell polyfunctionality was assessed by staining with mAbs to IFN-γ, TNF-α, and CD107a in a multi-color intracellular cytokine staining assay. To highlight the utility of the Mac-CD8 Polyfunctionality Assay, we assessed the effects of influenza infection on the ability of human macrophages to present antigen to CD8+ T cells. We found that influenza infection of human MDMs can alter the effector efficacy of MDMs to activate more CD8+ T cells with cytotoxic capacity. This has important implications for understanding how the virus-infected macrophages affect adaptive immunity at the site of infection. PMID:24312096

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

PubMed

Brinãs, Raymond P; Sundgren, Andreas; Sahoo, Padmini; Morey, Susan; Rittenhouse-Olson, Kate; Wilding, Greg E; Deng, Wei; Barchi, Joseph J

2012-08-15

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.

PubMed

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

2016-01-01

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

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Molecular Cloning and Sequence of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Cathepsin S gene

USDA-ARS?s Scientific Manuscript database

Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. This enzyme digests the invariant chain molecules so that antigenic peptides are able to load on the class II-associated invariant chain peptide of MHC. The complexes can subsequently be presented to the CD4 cell surface. In ...

Murine T-Cell Response to Native and Recombinant Protein Antigens of Rickettsia Tsutsugamushi

DTIC Science & Technology

1993-02-01

Wright, and J. Sadoff. 1985. 18-kilodalton protein of Mycobacterium leprae recognized by Immunoenzymatic analysis by monoclonal antibodies of bacte- Vo...determinants and closely resembles T-cell antigenic determinants, Rothbard and Taylor, by the GroEL homolog (65 kDa) of Mycobacterium tuberculo- analysis of...not be completely present in protein that is recognized by 20% of the mycobacterium - peptide 91-110. If this were the core of the antigenic deter

Sibling rivalry: competition between MHC class II family members inhibits immunity.

PubMed

Denzin, Lisa K; Cresswell, Peter

2013-01-01

Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

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

Structural and functional characterization of peptide-{beta}{sub 2}m fused HLA-A2/MART1{sub 27-35} complexes

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

Shen Chuanlai; Chang Chienchung; Zhang Jianqiong

The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-{beta}{sub 2}m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1{sub 27-35} complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher bindingmore » ability with HLA-A2/MART1{sub 27-35} complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.« less

T Cell Epitope Mimicry between Sjögren’s Syndrome Antigen A (SSA)/Ro60 and Oral, Gut, Skin and Vaginal Bacteria.

PubMed Central

Szymula, Agnieszka; Rosenthal, Jacob; Szczerba, Barbara M; Bagavant, Harini; Fu, Shu Man; Deshmukh, Umesh S.

2014-01-01

This study was undertaken to test the hypothesis that Sjogren’s syndrome Antigen A (SSA)/Ro60-reactive T cells are activated by peptides originating from oral and gut bacteria. T cell hybridomas generated from HLA-DR3 transgenic mice recognized 3 regions on Ro60, with core epitopes mapped to amino acids 228-238, 246-256 and 371-381. BLAST analysis identified several mimicry peptides, originating from human oral, intestinal, skin and vaginal bacteria, as well as environmental bacteria. Amongst these, a peptide from the von Willebrand factor type A domain protein (vWFA) from the oral microbe Capnocytphaga ochracea was the most potent activator. Further, Ro60-reactive T cells were activated by recombinant vWFA protein and whole E. coli expressing this protein. These results demonstrate that peptides derived from normal human microbiota can activate Ro60-reactive T cells. Thus, immune responses to commensal microbiota and opportunistic pathogens should be explored as potential triggers for initiating autoimmunity in SLE and Sjögren’s syndrome. PMID:24576620

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

PubMed

MacLachlan, Bruce J; Greenshields-Watson, Alexander; Mason, Georgina H; Schauenburg, Andrea J; Bianchi, Valentina; Rizkallah, Pierre J; Sewell, Andrew K; Fuller, Anna; Cole, David K

2017-02-06

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

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

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

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

2008-09-17

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

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

PubMed Central

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

2007-01-01

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

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides.

PubMed

Bowen, Anthony; Wear, Maggie P; Cordero, Radames J B; Oscarson, Stefan; Casadevall, Arturo

2017-01-13

Studies in the 1980s first showed that some natural antibodies were "catalytic" and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Monoclonal Antibody to Cryptococcus neoformans Glucuronoxylomannan Manifests Hydrolytic Activity for Both Peptides and Polysaccharides*

PubMed Central

Wear, Maggie P.; Cordero, Radames J. B.; Oscarson, Stefan

2017-01-01

Studies in the 1980s first showed that some natural antibodies were “catalytic” and able to hydrolyze peptide or phosphodiester bonds in antigens. Many naturally occurring catalytic antibodies have since been isolated from human sera and associated with positive and negative outcomes in autoimmune disease and infection. The function and prevalence of these antibodies, however, remain unclear. A previous study suggested that the 18B7 monoclonal antibody against glucuronoxylomannan (GXM), the major component of the Cryptococcus neoformans polysaccharide capsule, hydrolyzed a peptide antigen mimetic. Using mass spectrometry and Förster resonance energy transfer techniques, we confirm and characterize the hydrolytic activity of 18B7 against peptide mimetics and show that 18B7 is able to hydrolyze an oligosaccharide substrate, providing the first example of a naturally occurring catalytic antibody for polysaccharides. Additionally, we show that the catalytic 18B7 antibody increases release of capsular polysaccharide from fungal cells. A serine protease inhibitor blocked peptide and oligosaccharide hydrolysis by 18B7, and a putative serine protease-like active site was identified in the light chain variable region of the antibody. An algorithm was developed to detect similar sites present in unique antibody structures in the Protein Data Bank. The putative site was found in 14 of 63 (22.2%) catalytic antibody structures and 119 of 1602 (7.4%) antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, supports the notion that this activity is an important immunoglobulin function in host defense. The discovery of GXM hydrolytic activity suggests new therapeutic possibilities for polysaccharide-binding antibodies. PMID:27872188

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdolamir; Sarrami, Ramin; Nasiry, Habib; Sadeghizadeh, Majid

2005-01-01

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.

Dendritic cells pulsed with a tumor-specific peptide induce long-lasting immunity and are effective against murine intracerebral melanoma.

PubMed

Heimberger, Amy B; Archer, Gary E; Crotty, Laura E; McLendon, Roger E; Friedman, Allan H; Friedman, Henry S; Bigner, Darell D; Sampson, John H

2002-01-01

Dendritic cells (DCs) are specialized cells of the immune system that are capable of generating potent immune responses that are active even within the "immunologically privileged" central nervous system. However, immune responses generated by DCs have also been demonstrated to produce clinically significant autoimmunity. Targeting the epidermal growth factor receptor variant III (EGFRvIII), which is a mutation specific to tumor tissue, could eliminate this risk. The purpose of this study was to demonstrate that DC-based immunizations directed solely against this tumor-specific antigen, which is commonly found on tumors that originate within or metastasize to the brain, could be efficacious. C3H mice were vaccinated with DCs mixed with a keyhole limpet hemocyanin conjugate of the tumor-specific peptide, PEP-3, which spans the EGFRvIII mutation, or the random-sequence peptide, PEP-1, and were intracerebrally challenged with a syngeneic melanoma expressing a murine homologue of EGFRvIII. Systemic immunization with DCs mixed with PEP-3-keyhole limpet hemocyanin generated antigen-specific immunity. Among mice challenged with intracerebral tumors, this resulted in an approximately 600% increase in the median survival time (>300 d, P < 0.0016), relative to control values. Sixty-three percent of mice treated with DCs mixed with the tumor-specific peptide survived in the long term and 100% survived rechallenge with tumor, indicating that antitumor immunological memory was also induced. In a murine melanoma model, immunization with DCs mixed with tumor-specific peptide results in an antigen-specific immunological response that recognizes the EGFRvIII mutation, has potent antitumor efficacy against intracerebral tumors that express EGFRvIII, and results in long-lasting antitumor immunity.

Repurposing Ospemifene for Potentiating an Antigen-Specific Immune Response

PubMed Central

Kao, Chiao-Jung; Wurz, Gregory T.; Lin, Yi-Chen; Vang, Daniel P.; Phong, Brian; DeGregorio, Michael W.

2016-01-01

Objective Ospemifene, an estrogen receptor agonist/antagonist approved for treatment of dyspareunia and vaginal dryness in postmenopausal women, has potential new indications as an immune modulator. The overall objective of the present series of preclinical studies was to evaluate the immunomodulatory activity of ospemifene in combination with a peptide cancer vaccine. Methods Immune regulating effects, mechanism of action and structure activity relationships of ospemifene and related compounds were evaluated by examining expression of T cell activating cytokines in vitro, and antigen-specific immune response and cytotoxic T-lymphocyte activity in vivo. The effects of ospemifene (OSP) on the immune response to a peptide cancer vaccine (PV) were evaluated following chronic [control (n=22); OSP 50 mg/kg (n=16); PV (n=6); OSP+PV (n=11)], intermittent [control (n=10); OSP 10 and 50 mg/kg (n=11); PV (n=11); combination treatment (n=11 each dose)] and pretreatment [control; OSP 100 mg/kg; PV 100 µg; combination treatment (n=8 all groups)] ospemifene oral dosing schedules in a total of 317 mixed-sex tumor-bearing and non-tumor-bearing mice. Results The results showed that ospemifene induced expression of the key TH1 cytokines interferon gamma and interleukin-2 in vitro, which may be mediated by stimulating T cells through phosphoinositide 3-kinase and calmodulin signaling pathways. In combination with an antigen-specific peptide cancer vaccine, ospemifene increased antigen-specific immune response and increased cytotoxic T-lymphocyte activity in tumor-bearing and non-tumor-bearing mice. The pretreatment, intermittent, and chronic dosing schedules of ospemifene activate naïve T cells, modulate antigen-induced tolerance and reduce tumor-associated, pro-inflammatory cytokines, respectively. Conclusions Taken together, ospemifene’s dose response and schedule-dependent immune modulating activity offers a method of tailoring and augmenting the efficacy of previously failed antigen-specific cancer vaccines for a wide range of malignancies. PMID:27922937

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization

PubMed Central

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-01-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8+ T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-γ (IFN-γ) responses of almost 1% of CD8+ T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7–9 days, before detectable IFN-γ-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA+ CCR7− CD62L−) phenotype. The T-cell receptor Vβ analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness. PMID:19740333

Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization.

PubMed

Co, Mary Dawn T; Kilpatrick, Elizabeth D; Rothman, Alan L

2009-09-01

Management of yellow fever is focused on the prevention of illness by the use of the yellow fever virus (YFV) 17D vaccine. The role of neutralizing antibodies in protection is generally accepted with YFV-specific T cells likely contributing to the control of viral replication. We studied CD8(+) T-cell responses to four defined human leucocyte antigen-B35-restricted epitopes in YFV vaccine recipients as a model of the kinetics of cytotoxic T-lymphocyte responses to an acute human viral infection. Multiple features of these epitope-specific responses were analysed after vaccination including magnitude, cytokine production, phenotype and T-cell receptor repertoire. Peak peptide-specific interferon-gamma (IFN-gamma) responses of almost 1% of CD8(+) T cells were seen as early as 2 weeks post-vaccination; however, dominant responses varied between donors. Peptide-specific responses were still detectable at 54 months post-vaccination. Tetramer-positive cells, at high frequencies, were detected as early as 7-9 days, before detectable IFN-gamma-producing cells, suggesting a defect in the functional capacity of some antigen-specific cells early post-vaccination. The predominant memory phenotype of the tetramer-positive population was a differentiated effector (CD45RA(+) CCR7(-) CD62L(-)) phenotype. The T-cell receptor Vbeta analysis revealed a diverse oligoclonal repertoire in tetramer-positive T-cell populations in two individuals. These characteristics of the YFV-specific T-cell response could contribute to vaccine effectiveness.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

PubMed

Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

2017-09-15

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses. Copyright © 2017 American Society for Microbiology.

PD-1 signalling in CD4+ T cells restrains their clonal expansion to an immunogenic stimulus, but is not critically required for peptide-induced tolerance

PubMed Central

Konkel, Joanne E; Frommer, Friederike; Leech, Melanie D; Yagita, Hideo; Waisman, Ari; Anderton, Stephen M

2010-01-01

The ultimate outcome of T-cell recognition of peptide–major histocompatibility complex (MHC) complexes is determined by the molecular context in which antigen presentation is provided. The paradigm is that, after exposure to peptides presented by steady-state dendritic cells (DCs), inhibitory signals dominate, leading to the deletion and/or functional inactivation of antigen-reactive T cells. This has been utilized in a variety of models providing peptide antigen in soluble form in the absence of adjuvant. A co-inhibitory molecule of considerable current interest is PD-1. Here we show that there is the opportunity for the PD-1/PD-L1 interaction to function in inhibiting the T-cell response during tolerance induction. Using traceable CD4+ T-cell receptor (TCR) transgenic cells, together with a blocking antibody to disrupt PD-1 signalling, we explored the roles of PD-1 in the induction of tolerance versus a productive immune response. Intact PD-1 signalling played a role in limiting the extent of CD4+ T-cell accumulation in response to an immunogenic stimulus. However, PD-1 signalling was not required for either the induction, or the maintenance, of peptide-induced tolerance; a conclusion underlined by successful tolerance induction in TCR transgenic cells genetically deficient for PD-1. These observations contrast with the reported requirement for PD-1 signals in CD8+ T-cell tolerance. PMID:20113370

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

PubMed Central

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

2016-01-01

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

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants.

PubMed

Routsias, John G; Touloupi, Evgenia; Dotsika, Eleni; Moulia, Avrilia; Tsikaris, Vassilios; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Moutsopoulos, Haralampos M; Tzioufas, Athanasios G

2002-06-01

Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.

Molecular Pathways for Immune Recognition of Preproinsulin Signal Peptide in Type 1 Diabetes.

PubMed

Kronenberg-Versteeg, Deborah; Eichmann, Martin; Russell, Mark A; de Ru, Arnoud; Hehn, Beate; Yusuf, Norkhairin; van Veelen, Peter A; Richardson, Sarah J; Morgan, Noel G; Lemberg, Marius K; Peakman, Mark

2018-04-01

The signal peptide region of preproinsulin (PPI) contains epitopes targeted by HLA-A-restricted (HLA-A0201, A2402) cytotoxic T cells as part of the pathogenesis of β-cell destruction in type 1 diabetes. We extended the discovery of the PPI epitope to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles, revealing that four of six alleles present epitopes derived from the signal peptide region. During cotranslational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells, we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter associated with antigen processing (TAP), ER-luminal epitopes, which are TAP independent, each presented by different HLA class I molecules and N-terminal trimmed by ER aminopeptidase 1 for optimal presentation. In vivo, TAP expression is significantly upregulated and correlated with HLA class I hyperexpression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis. © 2018 by the American Diabetes Association.

Dynamic characterization of HLA-B*44 Alleles: A comparative molecular dynamics simulation study.

PubMed

Ozbek, Pemra

2016-06-01

Human Leukocyte Antigens (HLA) are highly polymorphic proteins that play a key role in the immune system. HLA molecule is present on the cell membrane of antigen-presenting cells of the immune system and presents short peptides, originating from the proteins of invading pathogens or self-proteins, to the T-cell Receptor (TCR) molecule of the T-cells. In this study, peptide-binding characteristics of HLA-B*44:02, 44:03, 44:05 alleles bound to three nonameric peptides were studied using molecular dynamics simulations. Polymorphisms among these alleles (Asp116Tyr and Asp156Leu) result in major differences in the allele characteristics. While HLA-B*44:02 (Asp116, Asp156) and HLA-B*44:03 (Asp116, Leu156) depend on tapasin for efficient peptide loading, HLA-B*44:05 (Tyr116, Asp156) is tapasin independent. On the other hand, HLA-B*44:02 and HLA-B*44:03 mismatch is closely related to transplant rejection and acute-graft-versus-host disease. In order to understand the dynamic characteristics, the simulation trajectories were analyzed by applying Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) calculations and hydrogen bonding analysis. Binding dynamics of the three HLA-B*44 alleles and peptide sequences are comparatively discussed. In general, peptide binding stability is found to depend on the peptide rather than the allele type for HLA-B*44 alleles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a.

PubMed Central

Turbyfill, K R; Joseph, S W; Oaks, E V

1995-01-01

The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection. PMID:7558301

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

Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.

PubMed

Chen, B P; Madrigal, A; Parham, P

1990-09-01

Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.

T-cell antigenic sites involved in myasthenia gravis: correlations with antibody titre and disease severity.

PubMed

Berrih-Aknin, S; Cohen-Kaminsky, S; Lepage, V; Neumann, D; Bach, J F; Fuchs, S

1991-02-01

We have evaluated the ability of eight synthetic peptides corresponding to selected regions of the alpha-subunit from human (H) or Torpedo (T) acetylcholine receptor (AChR) to stimulate proliferative responses of peripheral blood lymphocytes (PBL) and thymic cells from patients with Myasthenia Gravis (MG) in comparison to healthy controls. Using PBL, two of the peptides were most reactive: in the 40 myasthenic patients tested, peptide 169-181 (H) induced significant proliferative responses in 10 patients and peptide 351-368 (H) in five, while there was no response in any of the 34 healthy controls tested. Interestingly, clear associations between proliferation to peptides and clinical data were observed. Indeed, among responding patients, all presented thymic hyperplasia and most showed a high anti-AChR Ab titre and/or a severe form of the disease. In addition, responses to AChR cytoplasmic sequences were observed only in severely affected patients. Correlation with HLA-DR haplotype, sought in a subgroup of patients, indicated that response to 169-181 (H) is associated with HLA-DR5 in the patients presenting a high anti-AChR antibody titre. Using thymic lymphocytes, few responses were obtained with the human peptides, suggesting that the frequency of autoreactive cells is lower than in the blood. Similar to PBL, responses to peptides were observed only with lymphocytes isolated from hyperplastic thymuses. The correlations observed between responses to peptides and clinical parameters underline the pathophysiological relevance of our data and indicate that pathogenic and nonpathogenic T-cell antigenic sites involved in the anti-AChR response could be identified by this approach.

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

PubMed

Dahr, W; Knuppertz, G; Beyreuther, K; Moulds, J J; Moulds, M; Wilkinson, S; Capon, C; Fournet, B; Issitt, P D

1991-08-01

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

PubMed

Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael; Stryhn, Anette; Buus, Søren; Nielsen, Morten

2015-11-01

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

PubMed Central

Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J.; Calvo-Calle, J. Mauricio

2015-01-01

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B. PMID:26599878

Why do proteases mess up with antigen presentation by re-shuffling antigen sequences?

PubMed

Liepe, Juliane; Ovaa, Huib; Mishto, Michele

2018-04-30

The sequence of a large number of MHC-presented epitopes is not present as such in the original antigen because it has been re-shuffled by the proteasome or other proteases. Why do proteases throw a spanner in the works of our model of antigen tagging and immune recognition? We describe in this review what we know about the immunological relevance of post-translationally spliced epitopes and why proteases seem to have a second (dark) personality, which is keen to create new peptide bonds. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Dendrimer-conjugated peptide vaccine enhances clearance of Chlamydia trachomatis genital infection.

PubMed

Ganda, Ingrid S; Zhong, Qian; Hali, Mirabela; Albuquerque, Ricardo L C; Padilha, Francine F; da Rocha, Sandro R P; Whittum-Hudson, Judith A

2017-07-15

Peptide-based vaccines have emerged in recent years as promising candidates in the prevention of infectious diseases. However, there are many challenges to maintaining in vivo peptide stability and enhancement of peptide immunogenicity to generate protective immunity which enhances clearance of infections. Here, a dendrimer-based carrier system is proposed for peptide-based vaccine delivery, and shows its anti-microbial feasibility in a mouse model of Chlamydia trachomatis. Chlamydiae are the most prevalent sexually transmitted bacteria worldwide, and also the causal agent of trachoma, the leading cause of preventable infectious blindness. In spite of the prevalence of this infectious agent and the many previous vaccine-related studies, there is no vaccine commercially available. The carrier system proposed consists of generation 4, hydroxyl-terminated, polyamidoamine (PAMAM) dendrimers (G4OH), to which a peptide mimic of a chlamydial glycolipid antigen-Peptide 4 (Pep4, AFPQFRSATLLL) was conjugated through an ester bond. The ester bond between G4OH and Pep4 is expected to break down mainly in the intracellular environment for antigen presentation. Pep4 conjugated to dendrimer induced Chlamydia-specific serum antibodies after subcutaneous immunizations. Further, this new vaccine formulation significantly protected immunized animals from vaginal challenge with infectious Chlamydia trachomatis, and it reduced infectious loads and tissue (genital tract) damage. Pep4 conjugated to G4OH or only mixed with peptide provided enhanced protection compared to Pep4 and adjuvant (i.e. alum), suggesting a potential adjuvant effect of the PAMAM dendrimer. Combined, these results demonstrate that hydroxyl-terminated PAMAM dendrimer is a promising polymeric nanocarrier platform for the delivery of peptide vaccines and this approach has potential to be expanded to other infectious intracellular bacteria and viruses of public health significance. Copyright © 2017 Elsevier B.V. All rights reserved.

Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine.

PubMed

Hu, Yinin; Petroni, Gina R; Olson, Walter C; Czarkowski, Andrea; Smolkin, Mark E; Grosh, William W; Chianese-Bullock, Kimberly A; Slingluff, Craig L

2014-08-01

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4(+) T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8(+) T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund's adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4(+) and CD8(+) T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4(+) T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3281-295 (49 %) and tyrosinase386-406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8(+) T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4(+) and CD8(+) T cell responses against melanoma antigens. CD4(+) T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8(+) T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.

Serological profiling of the EBV immune response in Chronic Fatigue Syndrome using a peptide microarray.

PubMed

Loebel, Madlen; Eckey, Maren; Sotzny, Franziska; Hahn, Elisabeth; Bauer, Sandra; Grabowski, Patricia; Zerweck, Johannes; Holenya, Pavlo; Hanitsch, Leif G; Wittke, Kirsten; Borchmann, Peter; Rüffer, Jens-Ulrich; Hiepe, Falk; Ruprecht, Klemens; Behrends, Uta; Meindl, Carola; Volk, Hans-Dieter; Reimer, Ulf; Scheibenbogen, Carmen

2017-01-01

Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.

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

PubMed

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

2012-01-01

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

Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT

PubMed Central

Maecker, Holden T; Moon, James; Bhatia, Sonny; Ghanekar, Smita A; Maino, Vernon C; Payne, Janice K; Kuus-Reichel, Kristine; Chang, Jennie C; Summers, Amanda; Clay, Timothy M; Morse, Michael A; Lyerly, H Kim; DeLaRosa, Corazon; Ankerst, Donna P; Disis, Mary L

2005-01-01

Background Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. Results Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay. Conclusion We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems. PMID:16026627

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

PubMed Central

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

2002-01-01

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

Y2K+1 state-of-the-art on non-peptide phosphoantigens, a novel category of immunostimulatory molecules.

PubMed

Espinosa, E; Belmant, C; Sicard, H; Poupot, R; Bonneville, M; Fournié, J J

2001-07-01

Some human T cells are activated in vivo and in vitro by small non-peptide antigens, so-called phosphoantigens. Since their discovery in 1994, several reports have continuously documented novel members of this category of immunostimulatory molecules. This article reviews the current knowledge on their biochemical properties.

A Protective Vaccine against Chlamydia Genital Infection Using Vault Nanoparticles without an Added Adjuvant.

PubMed

Jiang, Janina; Liu, Guangchao; Kickhoefer, Valerie A; Rome, Leonard H; Li, Lin-Xi; McSorley, Stephen J; Kelly, Kathleen A

2017-01-19

Chlamydia trachomatis genital infection is the most common sexually transmitted bacterial disease, causing a significant burden to females due to reproductive dysfunction. Intensive screening and antibiotic treatment are unable to completely prevent female reproductive dysfunction, thus, efforts have become focused on developing a vaccine. A major impediment is identifying a safe and effective adjuvant which induces cluster of differentiation 4 (CD4) cells with attributes capable of halting genital infection and inflammation. Previously, we described a natural nanocapsule called the vault which was engineered to contain major outer membrane protein (MOMP) and was an effective vaccine which significantly reduced early infection and favored development of a cellular immune response in a mouse model. In the current study, we used another chlamydial antigen, a polymorphic membrane protein G-1 (PmpG) peptide, to track antigen-specific cells and evaluate, in depth, the vault vaccine for its protective capacity in the absence of an added adjuvant. We found PmpG-vault immunized mice significantly reduced the genital bacterial burden and histopathologic parameters of inflammation following a C. muridarum challenge. Immunization boosted antigen-specific CD4 cells with a multiple cytokine secretion pattern and reduced the number of inflammatory cells in the genital tract making the vault vaccine platform safe and effective for chlamydial genital infection. We conclude that vaccination with a Chlamydia -vault vaccine boosts antigen-specific immunities that are effective at eradicating infection and preventing reproductive tract inflammation.

T cell receptors used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms1

PubMed Central

Borbulevych, Oleg Y.; Santhanagopolan, Sujatha M.; Hossain, Moushumi; Baker, Brian M.

2011-01-01

T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Whereas DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity towards both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same antigens and the finding that TCR binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity. PMID:21795600

Presentation of peptides from Bacillus anthracis protective antigen on Tobacco Mosaic Virus as an epitope targeted anthrax vaccine.

PubMed

McComb, Ryan C; Ho, Chi-Lee; Bradley, Kenneth A; Grill, Laurence K; Martchenko, Mikhail

2015-11-27

The current anthrax vaccine requires improvements for rapidly invoking longer-lasting neutralizing antibody responses with fewer doses from a well-defined formulation. Designing antigens that target neutralizing antibody epitopes of anthrax protective antigen, a component of anthrax toxin, may offer a solution for achieving a vaccine that can induce strong and long lasting antibody responses with fewer boosters. Here we report implementation of a strategy for developing epitope focused virus nanoparticle vaccines against anthrax by using immunogenic virus particles to present peptides derived from anthrax toxin previously identified in (1) neutralizing antibody epitope mapping studies, (2) toxin crystal structure analyses to identify functional regions, and (3) toxin mutational analyses. We successfully expressed two of three peptide epitopes from anthrax toxin that, in previous reports, bound antibodies that were partially neutralizing against toxin activity, discovered cross-reactivity between vaccine constructs and toxin specific antibodies raised in goats against native toxin and showed that antibodies induced by our vaccine constructs also cross-react with native toxin. While protection against intoxication in cellular and animal studies were not as effective as in previous studies, partial toxin neutralization was observed in animals, demonstrating the feasibility of using plant-virus nanoparticles as a platform for epitope defined anthrax vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antigen-activated dendritic cells ameliorate influenza A infections

PubMed Central

Boonnak, Kobporn; Vogel, Leatrice; Orandle, Marlene; Zimmerman, Daniel; Talor, Eyal; Subbarao, Kanta

2013-01-01

Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections. PMID:23934125

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing.

PubMed

Morozov, Giora I; Zhao, Huaying; Mage, Michael G; Boyd, Lisa F; Jiang, Jiansheng; Dolan, Michael A; Venna, Ramesh; Norcross, Michael A; McMurtrey, Curtis P; Hildebrand, William; Schuck, Peter; Natarajan, Kannan; Margulies, David H

2016-02-23

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

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

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE PAGES

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.; ...

2016-02-11

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

T-Epitope Designer: A HLA-peptide binding prediction server.

PubMed

Kangueane, Pandjassarame; Sakharkar, Meena Kishore

2005-05-15

The current challenge in synthetic vaccine design is the development of a methodology to identify and test short antigen peptides as potential T-cell epitopes. Recently, we described a HLA-peptide binding model (using structural properties) capable of predicting peptides binding to any HLA allele. Consequently, we have developed a web server named T-EPITOPE DESIGNER to facilitate HLA-peptide binding prediction. The prediction server is based on a model that defines peptide binding pockets using information gleaned from X-ray crystal structures of HLA-peptide complexes, followed by the estimation of peptide binding to binding pockets. Thus, the prediction server enables the calculation of peptide binding to HLA alleles. This model is superior to many existing methods because of its potential application to any given HLA allele whose sequence is clearly defined. The web server finds potential application in T cell epitope vaccine design. http://www.bioinformation.net/ted/

Peptide vaccine immunotherapy biomarkers and response patterns in pediatric gliomas

PubMed Central

Müller, Sören; Agnihotri, Sameer; Shoger, Karsen E.; Myers, Max I.; Chaparala, Srilakshmi; Villanueva, Clarence R.; Chattopadhyay, Ansuman; Butterfield, Lisa H.; Okada, Hideho; Pollack, Ian F.

2018-01-01

Low-grade gliomas (LGGs) are the most common brain tumor affecting children. We recently reported an early phase clinical trial of a peptide-based vaccine, which elicited consistent antigen-specific T cell responses in pediatric LGG patients. Additionally, we observed radiologic responses of stable disease (SD), partial response (PR), and near-complete/complete response (CR) following therapy. To identify biomarkers of clinical response in peripheral blood, we performed RNA sequencing on PBMC samples collected at multiple time points. Patients who showed CR demonstrated elevated levels of T cell activation markers, accompanied by a cytotoxic T cell response shortly after treatment initiation. At week 34, patients with CR demonstrated both IFN signaling and Poly-IC:LC adjuvant response patterns. Patients with PR demonstrated a unique, late monocyte response signature. Interestingly, HLA-V expression, before or during therapy, and an early monocytic hematopoietic response were strongly associated with SD. Finally, low IDO1 and PD-L1 expression before treatment and early elevated levels of T cell activation markers were associated with prolonged progression-free survival. Overall, our data support the presence of unique peripheral immune patterns in LGG patients associated with different radiographic responses to our peptide vaccine immunotherapy. Future clinical trials, including our ongoing phase II LGG vaccine immunotherapy, should monitor these response patterns. PMID:29618666

Presentation of lipid antigens to T cells.

PubMed

Mori, Lucia; De Libero, Gennaro

2008-04-15

T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells

PubMed Central

Lichtenegger, Felix S.; Rothe, Maurine; Schnorfeil, Frauke M.; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus. PMID:29535740

Targeting LAG-3 and PD-1 to Enhance T Cell Activation by Antigen-Presenting Cells.

PubMed

Lichtenegger, Felix S; Rothe, Maurine; Schnorfeil, Frauke M; Deiser, Katrin; Krupka, Christina; Augsberger, Christian; Schlüter, Miriam; Neitz, Julia; Subklewe, Marion

2018-01-01

Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4 + and CD8 + T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.

The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells.

PubMed

Altomonte, M; Pucillo, C; Maio, M

1999-06-01

Besides their "classical" antigenic peptide-presenting activity, major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune and nonimmune cells. However, this "nonclassical" role and its functional consequences are still substantially overlooked. In this review, we will focus on these alternative functional properties of MHC class II antigens, to reawaken attention to their present and foreseeable immunobiologic and pathogenetic implications. The main issues that will be addressed concern 1) the role of MHC class II molecules as basic components of exchangeable oligomeric protein complexes with intracellular signaling ability; 2) the nonclassical functions of MHC class II antigens in immune cells; 3) the pathogenetic role of MHC class II antigens in inflammatory/autoimmune and infectious disease; and 4) the functional role of MHC class II antigens in solid malignancies.

Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

PubMed Central

Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

2013-01-01

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

PubMed Central

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L.; Wormald, Michael M.; Fast, Randy L.; Worsham, Patricia L.; Cote, Christopher K.; Amemiya, Kei; Dimitrov, Dimiter S.

2010-01-01

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans. PMID:20976274

Greek rheumatoid arthritis patients have elevated levels of antibodies against antigens from Proteus mirabilis.

PubMed

Christopoulos, Georgios; Christopoulou, V; Routsias, J G; Babionitakis, A; Antoniadis, C; Vaiopoulos, G

2017-03-01

Patients with rheumatoid arthritis (RA) from different ethnic groups present elevated levels of antibodies against Proteus mirabilis. This finding implicates P. mirabilis in the development of RA. The aim of this study was to investigate the importance of P. mirabilis in the etiopathogenesis of RA in Greek RA patients. In this study, 63 patients with RA and 38 healthy controls were included. Class-specific antibodies IgM, IgG, and IgA against three human cross-reactive and non-cross-reactive synthetic peptides from P. mirabilis-hemolysin (HpmB), urease C (UreC), and urease F (UreF)-were performed in all subjects, using the ELISA method. RA patients had elevated levels of IgM, IgG, and IgA antibodies against HpmB and UreC Proteus peptide which are significantly different compared to healthy controls: p = 0.005, p < 0.001, and p = 0.003 and p = 0.007, p = 0.002, and p < 0.001, correspondingly. Also, elevated levels of IgM, IgG, and IgA antibodies against the UreF Proteus peptide-which are non-cross-reactive with human tissue antigens-were observed and their significant difference compared to healthy controls (p = 0.007, p < 0.001, p < 0.001). Anti-peptide antibodies in RA patients showed a significant correlation with rheumatoid factors (Rf), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), especially when patients were divided into subgroups according to the receiving treatment. Greek RA patients present elevated levels of antibodies against P. mirabilis antigenic epitopes, such as in North European populations, albeit Greek RA patients presenting the cross-reaction antigen in a low percentage. These results indicate that P. mirabilis through the molecular mimicry mechanism leads to inflammation and damage of the joints in RA.

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

PubMed

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L; Wormald, Michael M; Fast, Randy L; Worsham, Patricia L; Cote, Christopher K; Amemiya, Kei; Dimitrov, Dimiter S

2010-10-13

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers

PubMed Central

Drake, Lisa A.; Drake, James R.

2016-01-01

MHC class II molecules present antigen-derived peptides to CD4 T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4 T cells. Motif 1 (M1) paired I-Ak class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2 mAb selectively binds M1 paired I-Ak class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. PMID:27148821

Systemically administered gp100 encoding DNA vaccine for melanoma using water-in-oil-in-water multiple emulsion delivery systems.

PubMed

Kalariya, Mayurkumar; Amiji, Mansoor M

2013-09-10

The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Engineering filamentous phage carriers to improve focusing of antibody responses against peptides.

PubMed

van Houten, Nienke E; Henry, Kevin A; Smith, George P; Scott, Jamie K

2010-03-02

The filamentous bacteriophage are highly immunogenic particles that can be used as carrier proteins for peptides and presumably other haptens and antigens. Our previous work demonstrated that the antibody response was better focused against a synthetic peptide if it was conjugated to phage as compared to the classical carrier, ovalbumin. We speculated that this was due, in part, to the relatively low surface complexity of the phage. Here, we further investigate the phage as an immunogenic carrier, and the effect reducing its surface complexity has on the antibody response against peptides that are either displayed as recombinant fusions to the phage coat or are chemically conjugated to it. Immunodominant regions of the minor coat protein, pIII, were removed from the phage surface by excising its N1 and N2 domains (Delta3 phage variant), whereas immunodominant epitopes of the major coat protein, pVIII, were altered by reducing the charge of its surface-exposed N-terminal residues (Delta8 phage variant). Immunization of mice revealed that the Delta3 variant was less immunogenic than wild-type (WT) phage, whereas the Delta8 variant was more immunogenic. The immunogenicity of two different peptides was tested in the context of the WT and Delta3 phage in two different forms: (i) as recombinant peptides fused to pVIII, and (ii) as synthetic peptides conjugated to the phage surface. One peptide (MD10) in its recombinant form produced a stronger anti-peptide antibody response fused to the WT carrier compared to the Delta3 phage carrier, and did not elicit a detectable anti-peptide response in its synthetic form conjugated to either phage carrier. This trend was reversed for a different peptide (4E10(L)), which did not produce a detectable anti-peptide antibody response as a recombinant fusion; yet, as a chemical conjugate to Delta3 phage, but not WT phage, it elicited a highly focused anti-peptide antibody response that exceeded the anti-carrier response by approximately 65-fold. The results suggest that focusing of the antibody response against synthetic peptides can be improved by decreasing the antigenic complexity of the phage surface. Copyright 2010 Elsevier Ltd. All rights reserved.

Eliciting Epitope-Specific CD8+ T Cell Response by Immunization with Microbial Protein Antigens Formulated with α-Galactosylceramide: Theory, Practice, and Protocols.

PubMed

Gilchuk, Pavlo; Knight, Frances C; Wilson, John T; Joyce, Sebastian

2017-01-01

CD8+ cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8+ T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. To induce a robust antigen-specific CD8+ T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8+ T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant.

Modular Design Features of a Peptide Amphiphile Micelle Vaccine Platform and Their Impact on an Immune Response

NASA Astrophysics Data System (ADS)

Barrett, John Christopher

Inducing a strong and specific immune response is the hallmark of a successful vaccine. Nanoparticles have emerged as promising vaccine delivery devices to discover and elicit immune responses. Modular platforms are attractive for their engineerability and broad potential applications. Fine-tuning a nanoparticle vaccine to create an immune response with specific antibody and other cellular responses is influenced by many factors such as shape, size and composition. Peptide amphiphile micelles are a unique biomaterials platform that can function as a modular vaccine delivery system, enabling control over many of these important factors. Peptide amphiphiles (PAs) consist of a hydrophilic peptide antigen conjugated to a hydrophobic lipid tail. The PAs then self-assemble into micelles, with the micelle characteristics determined by the chemical composition of the PA and micelle preparation methods. PA micelles contain a large design space, so it is important to have a basic understanding of how each design feature can affect the platform's interaction with the immune system. In this dissertation, the structure, composition, and biodistribution properties of PA micelles are evaluated for their ability to impact an immune response against a Group A Streptococcus B cell antigen (J8). Through structural design and physical characterization, micelles are shown to self-assemble into either short rod-like or long cylindrical shapes. Analyzing these shape effects on the immune response showed that cylindrical micelles induced higher antibody titers than rod-like micelles, providing evidence that the cylindrical micelle shape is important to induce immune responses and a possible mechanism of action. Shape was also seen to impact the activation profile of dendritic cells, B cells and T cells. Assembly into cylindrical micelles also stabilizes the secondary structure of peptide antigens, which may impact the immune response raised. In composition, the hydrophobic/hydrophilic interface of PA micelles enabled the precise entrapment of amphiphilic adjuvants which were found to not alter micelle formation or shape. These heterogeneous micelles significantly enhanced murine antibody responses when compared to animals vaccinated with non-adjuvanted micelles or soluble J8 peptide supplemented with a classical adjuvant. PAs were also shown to traffic more efficiently to the lymph node than free peptide. Characterization of these design features and their impact on an immune response provides a valuable foundation of knowledge to apply when expanding the peptide amphiphile micelle platform to other vaccine applications.

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.

PubMed

Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J

2011-03-01

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.

Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

PubMed Central

Chattopadhyay, Pratip K.; Melenhorst, J. Joseph; Ladell, Kristin; Gostick, Emma; Scheinberg, Philip; Barrett, A. John; Wooldridge, Linda; Roederer, Mario; Sewell, Andrew K.; Price, David A.

2008-01-01

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated tetrameric peptide-MHC class I (pMHCI) complexes in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where TCR-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer-binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidity, minimize background noise and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. PMID:18836993

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

HLA-B27.

PubMed

Bowness, Paul

2015-01-01

Possession of the human leukocyte antigen (HLA) class I molecule B27 is strongly associated with ankylosing spondylitis (AS), but the pathogenic role of HLA-B27 is unknown. Two broad theories most likely explain the role of HLA-B27 in AS pathogenesis. The first is based on the natural immunological function of HLA-B27 of presenting antigenic peptides to cytotoxic T cells. Thus, HLA-B27-restricted immune responses to self-antigens, or arthritogenic peptides, might drive immunopathology. B27 can also "behave badly," misfolding during assembly and leading to endoplasmic reticulum stress and autophagy responses. β2m-free B27 heavy chain structures including homodimers (B272) can also be expressed at the cell surface following endosomal recycling of cell surface heterotrimers. Cell surface free heavy chains and B272 bind to innate immune receptors on T, NK, and myeloid cells with proinflammatory effects. This review describes the natural function of HLA-B27, its disease associations, and the current theories as to its pathogenic role.

Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

PubMed

Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2014-01-01

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

PubMed

Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

2015-07-15

Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Primed tumor-reactive multifunctional CD62L+ human CD8+T-cells for immunotherapy

PubMed Central

Wölfl, Matthias; Merker, Katharina; Morbach, Henner; Van Gool, Stefaan W.; Eyrich, Matthias; Greenberg, Philip D.; Schlegel, Paul G.

2011-01-01

T-cell mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However ex vivo expansion of tumor-reactive T-cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T-cells. Here we show that when using highly purified naïve CD8+ T-cells, a single stimulation with peptide pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T-cells. Short-term expanded T-cells were tumor-reactive, multifunctional and retained a central memory-like phenotype (CD62L+, CCR7+, CD28+). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T-cells may therefore serve as a platform to target different malignancies accessible to immunotherapy. PMID:20972785

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

PubMed

Schofield, L; McConville, M J; Hansen, D; Campbell, A S; Fraser-Reid, B; Grusby, M J; Tachado, S D

1999-01-08

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

Immunization against Rabies with Plant-Derived Antigen

NASA Astrophysics Data System (ADS)

Modelska, Anna; Dietzschold, Bernard; Sleysh, N.; Fu, Zhen Fang; Steplewski, Klaudia; Hooper, D. Craig; Koprowski, Hilary; Yusibov, Vidadi

1998-03-01

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.

The Possible Mechanism of Idiosyncratic Lapatinib-Induced Liver Injury in Patients Carrying Human Leukocyte Antigen-DRB1*07:01

PubMed Central

Hirasawa, Makoto; Hagihara, Katsunobu; Okudaira, Noriko; Izumi, Takashi

2015-01-01

Idiosyncratic lapatinib-induced liver injury has been reported to be associated with human leukocyte antigen (HLA)-DRB1*07:01. In order to investigate its mechanism, interaction of lapatinib with HLA-DRB1*07:01 and its ligand peptide derived from tetanus toxoid, has been evaluated in vitro. Here we show that lapatinib enhances binding of the ligand peptide to HLA-DRB1*07:01. Furthermore in silico molecular dynamics analysis revealed that lapatinib could change the β chain helix in the HLA-DRB1*07:01 specifically to form a tightly closed binding groove structure and modify a large part of the binding groove. These results indicate that lapatinib affects the ligand binding to HLA-DRB1*07:01 and idiosyncratic lapatinib-induced liver injury might be triggered by this mechanism. This is the first report showing that the clinically available drug can enhance the binding of ligand peptide to HLA class II molecules in vitro and in silico. PMID:26098642

Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

USDA-ARS?s Scientific Manuscript database

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral vectored subunits, and synthetic peptides, most of which suffer from poor immunogenicity and are subject to degradation. For these reasons, efficient delivery systems and potent immunost...

Synthesis of antifungal vaccines by conjugation of β-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

PubMed

Cartmell, Jonathan; Paszkiewicz, Eugenia; Dziadek, Sebastian; Tam, Pui-Hang; Luu, Thanh; Sarkar, Susmita; Lipinski, Tomasz; Bundle, David R

2015-02-11

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (β-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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

2014-12-01

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