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Sample records for restricted bcl2-derived epitope

  1. A combination of epitope prediction and molecular docking allows for good identification of MHC class I restricted T-cell epitopes.

    PubMed

    Zhang, Xue Wu

    2013-08-01

    In silico identification of T-cell epitopes is emerging as a new methodology for the study of epitope-based vaccines against viruses and cancer. In order to improve accuracy of prediction, we designed a novel approach, using epitope prediction methods in combination with molecular docking techniques, to identify MHC class I restricted T-cell epitopes. Analysis of the HIV-1 p24 protein and influenza virus matrix protein revealed that the present approach is effective, yielding prediction accuracy of over 80% with respect to experimental data. Subsequently, we applied such a method for prediction of T-cell epitopes in SARS coronavirus (SARS-CoV) S, N and M proteins. Based on available experimental data, the prediction accuracy is up to 90% for S protein. We suggest the use of epitope prediction methods in combination with 3D structural modelling of peptide-MHC-TCR complex to identify MHC class I restricted T-cell epitopes for use in epitope based vaccines like HIV and human cancers, which should provide a valuable step forward for the design of better vaccines and may provide in depth understanding about activation of T-cell epitopes by MHC binding peptides.

  2. Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals.

    PubMed

    Yang, Junbao; James, Eddie; Roti, Michelle; Huston, Laurie; Gebe, John A; Kwok, William W

    2009-01-01

    Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159-171, 166-178, 449-461 and 1083-1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-gamma and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation.

  3. Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals

    PubMed Central

    Yang, Junbao; James, Eddie; Roti, Michelle; Huston, Laurie; Gebe, John A.

    2009-01-01

    Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159–171, 166–178, 449–461 and 1083–1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-γ and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation. PMID:19050106

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

    PubMed

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

    2015-01-22

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

  5. Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from proliferating cell nuclear antigen.

    PubMed

    Xu, Wei; Li, Hui-Zhong; Liu, Jun-Jie; Guo, Zhen; Zhang, Bao-Fu; Chen, Fei-Fei; Pei, Dong-Sheng; Zheng, Jun-Nian

    2011-02-01

    Peptide-based immunotherapy strategies appear promising as an approach to successfully induce an antitumor immune response and prolong survival in patients with various cancers. Protein antigens and their specific epitopes are formulation targets for anti-tumor vaccines. Bioinformatical approaches to predict major histocompatibility complex binding peptides can facilitate the resource-consuming effort of T cell epitope identification. Proliferating cell nuclear antigen including Ki-67 and PCNA, associated with the proliferation process of the cell, seems to be an attractive new target for tumor-specific immunotherapy. In this study, we predicted seven HLA-A*0201-restricted CTL candidate epitope of Ki-67 and eight epitope of PCNA by computer algorithm SYFPEITHI, BIMAS, and IEDB_ANN. Subsequently, biological functions of these peptides were tested by experiments in vitro. We found Ki-67((280-288)) (LQGETQLLV) had the strongest binding-affinity with HLA-A*0201. Further study revealed that Ki-67((280-288)) increased the frequency of IFN-γ-producing T cells compared to a negative peptide. Because Ki-67 was broadly expressed in most advanced malignant tumors, indicating a potential anti-tumor application in the future.

  6. Identification of Novel HLA-A*0201-Restricted CTL Epitopes in Chinese Vitiligo Patients

    PubMed Central

    Cui, Tingting; Yi, Xiuli; Guo, Sen; Zhou, Fubo; Liu, Ling; Li, Chunying; Li, Kai; Gao, Tianwen

    2016-01-01

    Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair. Recent studies suggested the key role of CD8+T lymphocytes for mediating immune response in vitiligo through melanocyte differentiation antigens, including tyrosinase, gp100 and MelanA/Mart-1. However, the specific epitopes of these auto-antigens are still unknown. In our study, we predicted the possible HLA-A*0201-restricted nonapeptides overlaying the full-length amino acid sequences of these three known antigens and investigated the lymphocytes reactivity to these nonapeptides by Elispot assay. In addition, we evaluated the abilities of these nonapeptides to activate CD8+T cells. We screened out 5 possible epitopes originated from tyrosinase and gp100, numbered P28, P41, P112, P118 and P119. Among these 5 epitopes, notably, P28 and P119 played the dominant role in activating CTLs, with a significant increase in proliferation rate and Interferon-γ (IFN-γ) production of CD8+T cells. Nevertheless, antigen-specific T cell reactivity was not detected in MelanA/Mart-1 peptides. Our studies identified two novel epitopes originated from proteins of gp100 and tyrosinase, which may have implications for the development of immunotherapies for vitiligo. PMID:27821860

  7. H5N1 strain-specific hemagglutinin CD4+ T cell epitopes restricted by HLA DR4.

    PubMed

    Yang, Junbao; Gebe, John A; Huston, Laurie; James, Eddie; Tan, Venus; Yue, Betty B; Nepom, Gerald T; Kwok, William W

    2009-06-12

    CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain-specific HLA class II restricted epitopes is essential for monitoring cellular strain-specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA(441-460) and H5HA(57-76) were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA(57-76) epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA(57-76) response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA(57-76) immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.

  8. H5N1 Strain-Specific Hemagglutinin CD4+ T cell Epitopes Restricted by HLA DR4

    PubMed Central

    Yang, Junbao; Gebe, John A.; Huston, Laurie; James, Eddie; Tan, Venus; Yue, Betty B.; Nepom, Gerald T.; Kwok, William W.

    2009-01-01

    CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain specific HLA class II restricted epitopes is essential for monitoring cellular strain specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA441-460 and H5HA57-76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA57-76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA57-76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA57-76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide. PMID:19446935

  9. HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

    PubMed Central

    Klein, Michèl R.; Hammond, Abdulrahman S.; Smith, Steve M.; Jaye, Assan; Lukey, Pauline T.; McAdam, Keith P. W. J.

    2002-01-01

    Few human CD8+ T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8+ T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha. PMID:11796635

  10. Identification of Mycobacterium tuberculosis PPE68-specific HLA-A*0201-restricted epitopes for tuberculosis diagnosis.

    PubMed

    Duan, Zhi-Liang; Li, Qiang; Wang, Sina; Chen, Xin-Yu; Liu, Hui-Fang; Chen, Bo-Kun; Li, De-Zhou; Huang, Xi; Wen, Jin-Sheng

    2015-06-01

    PPE68 is a Mycobacterium tuberculosis-specific protein which is absent from the vaccine strains of BCG. A panel of 14 PPE68-derived peptides predicted to bind to HLA-A*0201 was synthesized. The HLA-A*0201 restriction of these peptides was determined in T2 cell line and HLA-A*0201 transgenic mice. The specificity of peptides was assessed in pulmonary tuberculosis (TB) patients using IFN-γ enzyme-linked immunospot (ELISPOT) assay, and immunodominant peptides were further used to evaluate their diagnostic potential in HLA-A*0201-positive pulmonary TB patients. 13 out of 14 peptides were identified as high-affinity binders. Of these peptides, 12 peptides induced significant IFN-γ-secreting T cell response in transgenic mice and 9 peptides were efficiently recognized by peripheral blood mononuclear cells of 10 HLA-A*0201-positive TB patients. Four immunodominant HLA-A*0201-restricted epitopes (PPE68126-134, PPE68133-141, PPE68140-148, and PPE68148-156) were recognized by the most of 80 HLA-A*0201-positive TB patients (81, 86, 74, and 84 %, respectively). These epitopes may be used for a potential diagnosis of M. tuberculosis infection.

  11. HLA-A2-restricted cytotoxic T lymphocyte epitopes from human hepsin as novel targets for prostate cancer immunotherapy.

    PubMed

    Guo, J; Li, G; Tang, J; Cao, X-B; Zhou, Q-Y; Fan, Z-J; Zhu, B; Pan, X-H

    2013-09-01

    Hepsin is a type II transmembrane serine protease that is overexpressed in prostate cancer, and it is associated with prostate cancer cellular migration and invasion. Therefore, HPN is a biomarker for prostate cancer. CD8(+) T cells play an important role in tumour immunity. This study predicted and identified HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in human hepsin protein. HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: (1) a computer program generated predicted epitopes from the amino acid sequence of human hepsin; (2) an HLA-A2-binding assay detected the affinity of the predicted epitopes to the HLA-A2 molecule; (3) the primary T cell response against the predicted epitopes was stimulated in vitro; and (4) the induced CTLs towards different types of hepsin- or HLA-A2-expressing prostate cancer cells were detected. Five candidate peptides were identified. The effectors that were induced by human hepsin epitopes containing residues 229 to 237 (Hpn229; GLQLGVQAV), 268 to 276 (Hpn268; PLTEYIQPV) and 191 to 199 (Hpn199; SLLSGDWVL) effectively lysed LNCaP prostate cancer cells that were hepsin-positive and HLA-A2 matched. These peptide-specific CTLs did not lyse normal liver cells with low hepsin levels. Hpn229, Hpn268 and Hpn199 increased the frequency of IFN-γ-producing T cells compared with the negative peptide. These results suggest that the Hpn229, Hpn268 and Hpn199 epitopes are novel HLA-A2-restricted CTL epitopes that are capable of inducing hepsin-specific CTLs in vitro. Hpn229, Hpn268 and Hpn199 peptide-based vaccines may be useful for immunotherapy in patients with prostate cancer.

  12. Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

    PubMed Central

    Tan, Paul ThiamJoo; Heiny, A. T.; Miotto, Olivo; Salmon, Jerome; Marques, Ernesto T. A.; Lemonnier, Francois; August, J. Thomas

    2010-01-01

    Background The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. Methodology/Principal Findings HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. Conclusions/Significance Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus. PMID:20090904

  13. Identification of a dengue virus-specific HLA-A*0201-restricted CD8+ T cell epitope.

    PubMed

    Wen, Jinsheng; Duan, Zhiliang; Jiang, Lifang

    2010-04-01

    In this study, a combination of epitope-prediction programs and in vitro assays was used to identify dengue virus (DENV)-specific CD8(+) T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope-prediction programs. The IFN-gamma ELISpot results and the results of intracellular staining of IFN-gamma showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8(+) T cells. Furthermore, the results of the MHC-peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA-A*0201 molecules. The IFN-gamma ELISpot results and staining of intracellular IFN-gamma confirmed that this peptide could induce high-level CD8(+) T cell response in HLA-A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV-specific HLA-A*0201-restricted CD8(+) T cell epitope.

  14. Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins.

    PubMed

    Agallou, Maria; Athanasiou, Evita; Koutsoni, Olga; Dotsika, Eleni; Karagouni, Evdokia

    2014-01-01

    Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

  15. Identification of Two Novel HLA-A*0201-Restricted CTL Epitopes Derived from MAGE-A4

    PubMed Central

    Jia, Zheng-Cai; Ni, Bing; Huang, Ze-Min; Tian, Yi; Tang, Jun; Wang, Jing-Xue; Fu, Xiao-Lan; Wu, Yu-Zhang

    2010-01-01

    MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues except testis and placenta. MAGE-A antigens and their epitope peptides have been used in tumor immunotherapy trials. MAGE-A4 antigen is extensively expressed in various histological types of tumors, so it represents an attractive target for tumor immunotherapy. In this study, we predicted HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes of MAGE-A4, followed by peptide/HLA-A*0201 affinity and complex stability assays. Of selected four peptides (designated P1, P2, P3, and P4), P1 (MAGE-A4286-294, KVLEHVVRV) and P3 (MAGE-A4272-280, FLWGPRALA) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And the induced CTLs could lyse target cells in an HLA-A*0201-restricted fashion, demonstrating that the two peptides are HLA-A*0201-restricted CTL epitopes and could serve as targets for therapeutic antitumoral vaccination. PMID:21350607

  16. Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus

    PubMed Central

    Ma, Rui-xue; Cheng, Lin-feng; Ying, Qi-kang; Liu, Rong-rong; Ma, Tie-jun; Zhang, Xiao-xiao; Liu, Zi-yu; Zhang, Liang; Ye, Wei; Zhang, Fang-lin; Xu, Zhi-kai; Wang, Fang; Wu, Xing-an

    2016-01-01

    The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection. PMID:27933274

  17. 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.

  18. Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

    PubMed Central

    Hoppes, Rieuwert; Oostvogels, Rimke; Luimstra, Jolien J.; Wals, Kim; Toebes, Mireille; Bies, Laura; Ekkebus, Reggy; Rijal, Pramila; Celie, Patrick H. N.; Huang, Julie H.; Emmelot, Maarten E.; Spaapen, Robbert M.; Lokhorst, Henk; Schumacher, Ton N. M.; Mutis, Tuna; Ovaa, Huib

    2014-01-01

    Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients. PMID:25311806

  19. Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection

    PubMed Central

    Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry AF; Rothman, Alan L; Mathew, Anuja

    2014-01-01

    Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS126–34-specific CD8+ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS126–34-specific and other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8+ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation. PMID:23941420

  20. Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

    PubMed

    Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry A F; Rothman, Alan L; Mathew, Anuja

    2014-01-01

    Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.

  1. Identification of HLA-A*1101-restricted cytotoxic T lymphocyte epitopes derived from epidermal growth factor pathway substrate number 8

    PubMed Central

    Lu, Huifang; Tang, Baishan; He, Yanjie; Zhou, Weijun; Qiu, Jielei; Li, Yuhua

    2016-01-01

    Epidermal growth factor receptor pathway substrate 8 (EPS8) is critical in the proliferation, progression and metastasis of solid and hematological types of cancer, and thus constitutes an ideal target for cancer immunotherapy. The present study aimed to identify human leukocyte antigen (HLA)-A*1101-restricted cytotoxic T lymphocyte (CTL) epitopes from EPS8 and characterize their immunotherapeutic efficacy in vitro. Two computer-based algorithms were used to predict native EPS8 epitopes with potential high binding affinity to the HLA-A*1101 molecule, which is the HLA-A allele with the highest frequency in the Chinese population. The peptide-induced cytokine production from the CTLs was examined using enzyme-linked immunosorbent spot analysis. The cytotoxic effects on cancer cells by CTLs primed with the identified peptides were examined using flow cytometry. A total of five peptides, designated as P380, P70, P82, P30 and P529, presented with high affinity towards the HLA-A*1101 molecule. In response to stimulation by these five peptides, enhanced secretion of interferon-γ from the CTLs and increased cytolytic capabilities of the CTLs toward cancer cells were noted, with the most potent effects observed from the P380 peptide. Taken together, the present study identified five potential CTL epitopes from EPS8. Among these, P380 presented with the highest therapeutic efficacy in vitro. These peptides may benefit the development of EPS8-based immunotherapy for the treatment of HLA-A*1101-positive hematological malignancies. PMID:27840923

  2. HLA-DP, HLA-DQ, and HLA-DR-restricted epitopes in GRA5 of toxoplasma gondii strains

    NASA Astrophysics Data System (ADS)

    Haryati, S.; Sari, Y.; Prasetyo, A. A.; Sariyatun, R.

    2016-01-01

    The dense granular (GRA) proteins of Toxoplasma gondii(T. gondii) have been demonstrated as potential sources of T. gondii vaccine antigens. However, data of the GRA5 protein are limited. This study analyzed twenty-one complete GRA5 sequences of T. gondii GT1, RH, ME49, VEG, MAS, RUB, FOU, p89, VAND, and GAB2-2007-GAL-DOM2 strains to identify potential epitopes restricted by Major Histocompatibility Complex class II (MHC- II) molecules (human leukocyte antigen (HLA)-DP, HLA-DQ, and HLA-DR) in the protein. In all T. gondii strains, peptides positioned at amino acid (aa) 15-29, 16-30, 17-31, 18-32, 19-33, 83-97, 84-98, 86-100, 87-101, 89-103, and 90-104 were predicted to pose high affinity and binding with HLA-DRB1*0101, HLA-DRB1*0301 (DR17), HLA-DRB1*0401 (DR4Dw4), HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 (DR2b), and/or HLA-DRB5*0101. Considering the epitope's affinity, ligation strength, and hydrophilicity, LRLLRRRRRRAIQEE sequence (aa 90-104) restricted by HLA-DRB1*0101, HlA- DRB1*0301 (DR17), and HLA-DRB1*0401 (DR4Dw4) was considered as the most potential MHC-II epitope in GRA5 of T. gondii. These results would be useful for studies concerning in developing T. gondii vaccine and diagnostic method.

  3. Identification of protective Lassa virus epitopes that are restricted by HLA-A2.

    PubMed

    Botten, Jason; Alexander, Jeff; Pasquetto, Valerie; Sidney, John; Barrowman, Polly; Ting, Joey; Peters, Bjoern; Southwood, Scott; Stewart, Barbara; Rodriguez-Carreno, Maria P; Mothe, Bianca; Whitton, J Lindsay; Sette, Alessandro; Buchmeier, Michael J

    2006-09-01

    Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.

  4. Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

    PubMed Central

    Matsushita, Maiko; Otsuka, Yohei; Tsutsumida, Naoya; Tanaka, Chiaki; Uchiumi, Akane; Ozawa, Koji; Suzuki, Takuma; Ichikawa, Daiju; Aburatani, Hiroyuki; Okamoto, Shinichiro; Kawakami, Yutaka; Hattori, Yutaka

    2016-01-01

    The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients. PMID:26784514

  5. Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

    PubMed

    Günther, Patrick S; Peper, Janet K; Faist, Benjamin; Kayser, Simone; Hartl, Lena; Feuchtinger, Tobias; Jahn, Gerhard; Neuenhahn, Michael; Busch, Dirk H; Stevanović, Stefan; Dennehy, Kevin M

    2015-09-01

    Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

  6. Analysis of a subclass-restricted HIV-1 gp41 epitope by omission peptides.

    PubMed Central

    Mathiesen, T; Chiodi, F; Broliden, P A; Albert, J; Houghten, R A; Utter, G; Wahren, B; Norrby, E

    1989-01-01

    To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses. PMID:2472353

  7. Identification of conserved subdominant HIV Type 1 CD8(+) T Cell epitopes restricted within common HLA Supertypes for therapeutic HIV Type 1 vaccines.

    PubMed

    Karlsson, Ingrid; Kløverpris, Henrik; Jensen, Kristoffer Jarlov; Stryhn, Anette; Buus, Søren; Karlsson, Annika; Vinner, Lasse; Goulder, Philip; Fomsgaard, Anders

    2012-11-01

    The high HIV-1 prevalence, up to 4.6% in Guinea-Bissau, West Africa, makes it a relevant location for testing of therapeutic vaccines. With the aim of performing a clinical study in Guinea-Bissau, after first testing the vaccine for safety in Denmark, Europe, we here describe the design of a universal epitope peptide-based T cell vaccine with relevance for any geographic locations. The two major obstacles when designing such a vaccine are the high diversities of the HIV-1 genome and of the human major histocompatibility complex (MHC) class I. We selected 15 CD8-restricted epitopes predicted from conserved regions of HIV-1 that were subdominant (i.e., infrequently targeted) within natural infections. Moreover, the epitopes were predicted to be restricted to at least one of the five common HLA supertypes (HLA-A01, A02, A03, B07, and B44). Here, we validated the resulting peptide-specific, HLA-restricted T cell specificities using peptide-MHC class I tetramer labeling of CD8(+) T cells from HIV-1-infected individuals. The selected vaccine epitopes are infrequently targeted in HIV-1-infected individuals from both locations. Moreover, we HLA-typed HIV-1-infected individuals and demonstrated that the selected vaccine epitopes, when targeted, are restricted to the five most common HLA supertypes at both locations. Thus, the HLA supertype-directed approach achieved HLA coverage of 95% and 100% of the examined cohorts in Guinea-Bissau and Denmark, respectively. In conclusion, the selected vaccine epitopes match the host populations and HIV-1 strains of these two distant geographic regions, justifying clinical testing in both locations.

  8. MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination.

    PubMed

    Tang, S T; Wang, M; Lamberth, K; Harndahl, M; Dziegiel, M H; Claesson, M H; Buus, S; Lund, O

    2008-01-01

    It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-gamma release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity of K(D) less than or equal to 5 nM, whereas no T cell responses were detected against 60 peptides with an HLA affinity of K(D) more than 5 nM. All epitopes were fully conserved in seven variola, vaccinia and cowpox strains. Knowledge of the long-term response to smallpox vaccination may lead to a better understanding of poxvirus immunity and may aid in the development of new improved vaccines and diagnostic tools.

  9. Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

    PubMed Central

    Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

    2013-01-01

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

  10. Novel HLA-B27-restricted epitopes from Chlamydia trachomatis generated upon endogenous processing of bacterial proteins suggest a role of molecular mimicry in reactive arthritis.

    PubMed

    Alvarez-Navarro, Carlos; Cragnolini, Juan J; Dos Santos, Helena G; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A

    2013-09-06

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na(+)-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27(+) cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330-338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211-223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211-223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.

  11. Chicken antibody against a restrictive epitope of prion protein distinguishes normal and abnormal prion proteins.

    PubMed

    Miyamoto, Kazuyoshi; Kimura, Sota; Nakamura, Naoto; Yokoyama, Takashi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

    2007-10-01

    Recently, we reported the application of a recombinant chicken IgY monoclonal antibody, Ab3-15, against mammalian prion protein (PrP), for the diagnosis of bovine spongiform encephalopathy in cattle. In this study, we have characterized a soluble, single-chain variable fragment (scFv) form of this antibody, sphAb3-15 using brain homogenates from mice. This sphAb3-15 antibody recognized denatured forms of both PrP(C) and PrP(Sc), and PrP(Sc) after PK-treatment, on Western blotting. In sandwich ELISAs, on dot blots and by immunoprecipitation, sphAb3-15 efficiently bound to PrP from normal brain homogenates, but weakly bound PrP from scrapie-infected brain homogenates. These results suggest that sphAb3-15 selectively recognizes PrP(C) under native conditions and that the epitope recognized by sphAb3-15 may undergo conformational changes during the conversion of PrP(C) into PrP(Sc).

  12. Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.

    PubMed

    Skora, Andrew D; Douglass, Jacqueline; Hwang, Michael S; Tam, Ada J; Blosser, Richard L; Gabelli, Sandra B; Cao, Jianhong; Diaz, Luis A; Papadopoulos, Nickolas; Kinzler, Kenneth W; Vogelstein, Bert; Zhou, Shibin

    2015-08-11

    Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.

  13. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen

    PubMed Central

    1991-01-01

    The absence of readily manipulable experimental systems to study the cytotoxic T lymphocyte (CTL) response against hepatitis B virus (HBV) antigens has thus far precluded a definitive demonstration of the role played by this response in the pathogenesis of liver cell injury and viral clearance during HBV infection. To circumvent the problem that HBV infection of human cells in vitro for production of stimulator/target systems for CTL analysis is not feasible, a panel of 22 overlapping synthetic peptides covering the entire amino acid sequence of the HBV core (HBcAg) and e (HBeAg) antigens were used to induce and to analyze the HBV nucleocapsid-specific CTL response in nine patients with acute hepatitis B, six patients with chronic active hepatitis B, and eight normal controls. By using this approach, we have identified an HLA-A2-restricted CTL epitope, located within the NH2- terminal region of the HBV core molecule, which is shared with the e antigen and is readily recognized by peripheral blood mononuclear cells from patients with self-limited acute hepatitis B but less efficiently in chronic HBV infection. Our study provides the first direct evidence of HLA class I-restricted T cell cytotoxicity against HBV in humans. Furthermore, the different response in HBV-infected subjects who successfully clear the virus (acute patients) in comparison with patients who do not succeed (chronic patients) suggests a pathogenetic role for this CTL activity in the clearance of HBV infection. PMID:1720813

  14. Identification and enhancement of HLA-A2.1-restricted CTL epitopes in a new human cancer antigen-POTE.

    PubMed

    Huang, Yi-Hsiang; Terabe, Masaki; Pendleton, C David; Stewart Khursigara, Deborah; Bera, Tapan K; Pastan, Ira; Berzofsky, Jay A

    2013-01-01

    Identification of CD8(+) T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL) epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA) substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272-280 and POTE 323-331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9), POTE 553-1Y (with tyrosine at position 1) and POTE 323-3F (with phenylalanine at position 3) conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F) induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2(+) human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

  15. Identification of HLA-DRB1*1501-restricted T-cell epitopes from prostate-specific antigen.

    PubMed

    Klyushnenkova, Elena N; Link, Jason; Oberle, Warren T; Kodak, James; Rich, Cathleen; Vandenbark, Arthur A; Alexander, Richard B

    2005-04-15

    The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate tissue is very attractive because prostate is not a vital organ beyond the reproductive years. CD4 T cells play an important role in the development of antitumor immune responses, yet the identification of naturally processed MHC Class II-restricted epitopes derived from prostate differentiation antigens has not been described. To facilitate the search for prostate-specific antigen (PSA)-derived MHC class II-restricted peptides, we immunized mice transgenic for HLA-DRB1*1501 with human PSA and showed a robust dose-dependent immune response to the antigen. Screening a library of overlapping 20-mer peptides that span the entire PSA sequence identified two 20-mer peptides, PSA(171-190) and PSA(221-240), which were responsible for this reactivity. Immunization of DR2b transgenic mice with these peptides induced specific responses to the peptide and whole PSA. Identified peptides were used to stimulate CD4 T cells from HLA-DRB1*1501+ patients with a rare condition, granulomatous prostatitis, and who seem to have a preexisting immune response directed against the prostate gland. We previously showed a linkage of granulomatous prostatitis to HLA-DRB1*1501, suggesting that this disease may have an autoimmune etiology. Peptide-specific CD4 T-cell lines were generated from the peripheral blood of these patients as well as one patient with prostate cancer. These lines also recognized whole, processed PSA in the context of HLA-DRB1*1501. This study will be instrumental in understanding the interaction between circulating self-reactive T cells, organ-specific autoimmunity, and antitumor immune response. The use of these peptides for the immunotherapy of prostate cancer is under investigation.

  16. Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

    NASA Astrophysics Data System (ADS)

    Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

    2017-02-01

    Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.

  17. Immunogenicity and antigenicity of immunoglobulins. XII. Intact light chain and heavy chain isotype-restricted Vk-associated epitopes.

    PubMed

    Walker, M; Hardie, D; Lowe, J; Ling, N R; De Lange, G; Jefferis, R

    1985-06-01

    Immunization with intact IgG has allowed the isolation of four hybridomas producing antibodies recognizing epitopes expressed within subpopulations of human kappa light chains unrelated to known polymorphisms (Km) and previously defined V-region subgroups. The V-region-associated epitopes recognized are conformation-dependent, being expressed on intact light chain but not on isolated VK or CK fragments. The frequency of expression within paraprotein panels of different heavy chain isotypes varied between individual antibodies. An epitope recognized by B2A6, expressed by greater than 85% IgGK paraproteins, was not represented in 16 IgM paraproteins tested, suggesting that association of VK with mu chains does not result in display of the epitope recognized, or alternatively, that selective association between VK and CH gene products occurs. These data contrast with the reactivity of other McAb for CK epitopes which were reactive with isolated CK fragments, and for all kappa-bearing paraproteins, regardless of heavy chain isotypes.

  18. CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model.

    PubMed

    Chhabra, Arvind; Yang, Lili; Wang, Pin; Comin-Anduix, Begoña; Das, Raja; Chakraborty, Nitya G; Ray, Swagatam; Mehrotra, Shikhar; Yang, Haiguang; Hardee, Cinnamon L; Hollis, Roger; Dorsky, David I; Koya, Richard; Kohn, Donald B; Ribas, Antoni; Economou, James S; Baltimore, David; Mukherji, Bijay

    2008-07-15

    Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.

  19. CD4+CD25− T cells transduced to express MHC class I-restricted epitope specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model

    PubMed Central

    Chhabra, Arvind; Yang, Lili; Wang, Pin; Comin-Anduix, Begoña; Das, Raja; Chakraborty, Nitya G.; Ray, Swagatam; Mehrotra, Shikhar; Yang, Haiguang; Hardee, Cinnamon L.; Hollis, Roger; Dorsky, David I.; Koya, Richard; Kohn, Donald B.; Ribas, Antoni; Economou, James S.; Baltimore, David; Mukherji, Bijay

    2009-01-01

    Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4+ T cells. Considering the difficulties in simultaneously engaging CD4+ and CD8+ T cells in tumor immunotherapy -- especially in an antigen specific manner -- “redirecting” CD4+ T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope specific T cell receptors (TCR) in CD4+ T cells has emerged as a strategic consideration. Such TCR engineered CD4+ T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have carried out a critical examination of functional characteristics of CD4+ T cells engineered to express the α and β chains of a high functional avidity TCR specific for the melanoma epitope, MART-127–35 (M1), as a prototypic human tumor antigen system. We found that unpolarized CD4+CD25− T cells engineered to express the M1 TCR selectively synthesize Th1 cytokines and exhibit a potent antigen-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8+ CTL. Such TCR engineered CD4+ T cells, therefore, might be useful in clinical immunotherapy. PMID:18606658

  20. Functional and Structural Characterization of a Novel HLA-DRB1*04:01-Restricted α-Enolase T Cell Epitope in Rheumatoid Arthritis

    PubMed Central

    Gerstner, Christina; Dubnovitsky, Anatoly; Sandin, Charlotta; Kozhukh, Genadiy; Uchtenhagen, Hannes; James, Eddie A.; Rönnelid, Johan; Ytterberg, Anders Jimmy; Pieper, Jennifer; Reed, Evan; Tandre, Carolina; Rieck, Mary; Zubarev, Roman A.; Rönnblom, Lars; Sandalova, Tatyana; Buckner, Jane H.; Achour, Adnane; Malmström, Vivianne

    2016-01-01

    Antibodies to citrullinated proteins, common in rheumatoid arthritis (RA) patients, are strongly associated to a specific set of HLA-DR alleles including HLA-DRB1*04:01, *04:04, and *01:01. Here, we first demonstrate that autoantibody levels toward the dominant citrullinated B cell epitope from α-enolase are significantly elevated in HLA-DRB1*04:01-positive RA patients. Furthermore, we identified α-enolase-derived T cell epitopes and demonstrated that native and citrullinated versions of several peptides bind with different affinities to HLA-DRB1*04:01, *04:04, and *01:01. The citrulline residues in the eight identified peptides are distributed throughout the entire length of the presented epitopes and more specifically, localized at peptide positions p-2, p2, p4, p6, p7, p10, and p11. Importantly, in contrast to its native version peptide 26 (TSKGLFRAAVPSGAS), the HLA-DRB1*04:01-restricted citrullinated peptide Cit26 (TSKGLFCitAAVPSGAS) elicited significant functional T cell responses in primary cells from RA patients. Comparative analysis of the crystal structures of HLA-DRB1*04:01 in complex with peptide 26 or Cit26 demonstrated that the posttranslational modification did not alter the conformation of the peptide. And since citrullination is the only structural difference between the two complexes, this indicates that the neo-antigen Cit26 is recognized by T cells with high specificity to the citrulline residue. PMID:27895642

  1. Flavivirus-cross-reactive, HLA-DR15-restricted epitope on NS3 recognized by human CD4+ CD8- cytotoxic T lymphocyte clones.

    PubMed

    Kurane, I; Okamoto, Y; Dai, L C; Zeng, L L; Brinton, M A; Ennis, F A

    1995-09-01

    The role of flavivirus-cross-reactive T lymphocytes in recovery from and pathogenesis of flavivirus infections is not known. In the present paper, we have defined a flavivirus-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK4 and JK43. The T cell clones were established from the peripheral blood T lymphocytes of a dengue-4-immune donor, using a limiting-dilution method with dengue-4 antigen. These two T cell clones were cross-reactive for dengue virus types 1, 2, 3 and 4, yellow fever virus and West Nile virus, and recognized NS3 protein. The smallest synthetic peptide recognized by these T cell clones was an identical 9 amino acid peptide which contains amino acids 146 to 154 (VIGLYGNGV) of dengue-4 NS3. HLA-DR15 was the restriction allele for recognition of this epitope by JK4 and JK43. JK4 and JK43 both used T cell receptor V alpha 8, but JK4 used V beta 8 and JK43 used V beta 2. This result indicates that this epitope is recognized by two flavivirus-cross-reactive CD4+ T cell clones which originated from different T cells in vivo.

  2. Identificaiton of Novel Immunogenic Human Papillomavirus Type 16 E7-Specific Epitopes Restricted to HLA-A*33;03 for Cervical Cancer Immunotherapy

    PubMed Central

    Kim, Sunghoon; Chung, Hye Won; Kong, Hoon Young

    2017-01-01

    Purpose To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. Materials and Methods We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a 51Cr release assay with SNU1299 cells. Results Among the fourteen 15-amino acid peptides, E749-63 (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E749-63 showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E749-63, E750-59 (AHYNIVTFCC), and E752-61 (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E750-59- and E752-61-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. Conclusion We identified E750-59 and E752-61 as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer. PMID:27873494

  3. Identification and immunogenicity of two new HLA-A*0201-restricted CD8+ T-cell epitopes on dengue NS1 protein.

    PubMed

    Tian, Jiang; Zeng, Gucheng; Pang, Xianwu; Liang, Mifang; Zhou, Junmei; Fang, Danyun; Liu, Yan; Li, Dexin; Jiang, Lifang

    2012-04-01

    Immunopathogenesis of dengue virus (DEN) infection remains poorly studied. Identification and characterization of human CD8(+) T-cell epitopes on DEN are necessary for a better understanding of the immunopathogenesis of dengue infection and would facilitate the development of immunotherapy and vaccines to protect from dengue infection. Here, we identified two new HLA-A*0201-restricted CD8(+) T-cell epitopes, DEN-4 NS1(990)(-998) and DEN-4 NS1(997)(-1005) that are conserved in three or four major DEN serotypes, respectively. Unexpectedly, we found that immunization of HLA-A*0201 transgenic mice with DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of tumor necrosis factor (TNF)-α and IFN-γ, two important pro-inflammatory molecules that are hard to be detected directly without in vitro antigenic re-stimulation. Importantly, we demonstrated that CD8(+) T cells specifically activated by DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of perforin. Furthermore, we observed that DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005)-specific CD8(+) T cells capable of producing large amounts of perforin, TNF-α and IFN-γ preferentially displayed CD27(+)CD45RA(-), but not CD27(-)CD45RA(+), phenotypes. This study, therefore, suggested the importance of synergistic effects of pro-inflammatory cytokines and cytotoxic molecules which were produced by dengue-specific CD8(+) T cells in immunopathogenesis or anti-dengue immunity during dengue infection.

  4. Melan-A/MART-1(51-73) represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4(+) T cells.

    PubMed

    Zarour, H M; Kirkwood, J M; Kierstead, L S; Herr, W; Brusic, V; Slingluff, C L; Sidney, J; Sette, A; Storkus, W J

    2000-01-04

    The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8(+) cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4(+) T cells. The Melan-A/MART-1(51-73) peptide was able to induce the in vitro expansion of specific CD4(+) T cells derived from normal DR4(+) donors or from DR4(+) patients with melanoma when pulsed onto autologous dendritic cells. CD4(+) responder T cells specifically produced IFN-gamma in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-1(51-73) peptide and DR4(+) melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4(+) T cell immunoreactivity against the Melan-A/MART-1(51-73) peptide typically coexisted with a high frequency of anti-Melan-A/MART-1(27-35) reactive CD8(+) T cells in freshly isolated blood harvested from HLA-A2(+)/DR4(+) patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4(+) T cell responses against melanoma in vivo.

  5. Analysis of TCR antagonism and molecular mimicry of an HLA-A0201-restricted CTL epitope in primary biliary cirrhosis.

    PubMed

    Kita, Hiroto; Matsumura, Shuji; He, Xiao-Song; Ansari, Aftab A; Lian, Zhe-Xiong; Van de Water, Judy; Coppel, Ross L; Kaplan, Marshall M; Gershwin, M Eric

    2002-10-01

    Although the etiology and mechanism of primary biliary cirrhosis (PBC) is unknown, growing evidence suggests a major role for T cells. We have recently identified the first CD8 T-cell epitope, amino acid 159-167 of the E2 component of pyruvate dehydrogenase complexes (PDC-E2). To seek for analogue peptide-antagonizing effector function of CTLs specific for this autoantigen, we examined the effector functions of the PDC-E2-specific CTLs against alanine substituted peptides. Furthermore, because molecular mimicry has been postulated as a possible cause of initiating PBC, we carried out studies aimed at identifying naturally occurring peptides for the 159-167 peptide of PDC-E2 that may serve as agonists. An alanine substitution at position 5 of this epitope significantly reduced peptide-specific effector functions of CTLs. Moreover, this analogue peptide inhibited effector functions of the CTLs to the prototype peptide, including cytotoxicity and IFN-gamma production. We also identified a peptide derived from Pseudomonas aeruginosa, which showed a higher binding affinity to the HLA-A*0201 than the prototype peptide. This homologous peptide was recognized by CTLs specific for the prototype epitope on PDC-E2. In conclusion, a modification of the immunodominant autoepitope can be utilized to manipulate the CD8 T-cell responses against the autoantigen PDC-E2. Our finding also supports the thesis that molecular mimicry may be implicated in the initiation of the autoreactive CD8 T-cell responses and has implications for the use of such peptides for immunotherapy.

  6. Identification of human leukocyte antigen-A24-restricted epitope peptides derived from gene products upregulated in lung and esophageal cancers as novel targets for immunotherapy.

    PubMed

    Suda, Takako; Tsunoda, Takuya; Daigo, Yataro; Nakamura, Yusuke; Tahara, Hideaki

    2007-11-01

    For the development of cancer vaccine therapies, we have searched for possible epitope peptides that can elicit cytotoxic T lymphocytes (CTL) to the TTK protein kinase (TTK), lymphocyte antigen 6 complex locus K (LY6K) and insulin-like growth factor (IGF)-II mRNA binding protein 3 (IMP-3), which were previously identified to be transactivated in the majority of lung and esophageal cancers. We screened 31, 17 and 17 candidate human leukocyte antigen (HLA)-A*2402-binding peptides to parts of TTK, LY6K and IMP-3, respectively. As a result, we successfully established strong CTL clones stimulated by TTK-567 (SYRNEIAYL), LY6K-177 (RYCNLEGPPI) and IMP-3-508 (KTVNELQNL) that have specific cytotoxic activities against the HLA-A24-positive target cells pulsed with the candidate peptides. Subsequent analysis of the CTL clones also revealed their cytotoxic activities against lung and esophageal tumor cells that endogenously express TTK, LY6K or IMP-3. A cold target inhibition assay further confirmed that the CTL cell clones specifically recognized the MHC class I–peptide complex. Our results strongly imply that TTK, LY6K and IMP-3 are novel tumor-associated antigens recognized by CTL, and TTK-567 (SYRNEIAYL), LY6K-177 (RYCNLEGPPI) and IMP-3-508 (KTVNELQNL) are HLA-A24-restricted epitope peptides that can induce potent and specific immune responses against lung and esophageal cancer cells expressing TTK, LY6K and IMP-3.

  7. HLA-DR3 restricted T cell epitope mimicry in induction of autoimmune response to lupus-associated antigen SmD

    PubMed Central

    Deshmukh, Umesh S; Sim, Davis L; Dai, Chao; Kannapell, Carol J; Gaskin, Felicia; Rajagopalan, Govindarajan; David, Chella S; Fu, Shu Man

    2012-01-01

    Although systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder, HLA-D is the most dominant genetic susceptibility locus. This study was undertaken to investigate the hypothesis that microbial peptides bind HLA-DR3 and activate T cells reactive with lupus autoantigens. Using HLA-DR3 transgenic mice and lupus-associated autoantigen SmD protein, SmD79–93 was identified to contain a dominant HLA-DR3 restricted T cell epitope. This T cell epitope was characterized by using a T-T hybridoma, C1P2, generated from SmD immunized HLA-DR3 transgenic mouse. By pattern search analysis, 20 putative mimicry peptides (P2–P21) of SmD79–93, from microbial and human origin were identified. C1P2 cells responded to SmD, SmD79–93 and a peptide (P20) from Vibro cholerae. Immunization of HLA-DR3 mice with P20 induced T cell responses and IgG antibodies to SmD that were not cross-reactive with the immunogen. A T-T hybridoma, P20P1, generated from P20 immunized mice, not only responded to P20 and SmD79–93, but also to peptides from Streptococccus agalactiae (P17) and human-La related protein (P11). These three T cell mimics (P20, P11 and P17) induced diverse and different autoantibody response profiles. Our data demonstrates for the first time molecular mimicry at T cell epitope level between lupus-associated autoantigen SmD and microbial peptides. Considering distinct autoreactive T cell clones, activated by different microbial peptides, molecular mimicry at T cell epitope level can be an important pathway for the activation of autoreactive T cells resulting in the production of autoantibodies. In addition, the novel findings reported herein may have significant implications in the pathogenesis of SLE. PMID:21868195

  8. HIV-2 Surveillance with Next-Generation Sequencing Reveals Mutations in a Cytotoxic Lymphocyte-Restricted Epitope Involved in Long-Term Nonprogression.

    PubMed

    Yamaguchi, Julie; Brennan, Catherine A; Alessandri-Gradt, Elodie; Plantier, Jean-Christophe; Cloherty, Gavin A; Berg, Michael G

    2017-04-01

    HIV-2 exhibits a natural history of infection distinct from HIV-1. Primarily found in West Africa and in only 10%-20% of HIV infections in this region, patients with HIV-2 typically exhibit a slower progression to AIDS, lower viral loads, and decreased rates of transmission. Here, we used next-generation sequencing to determine the sequence and phylogenetic classification of nine HIV-2 genomes. We identified a patient with a series of mutations in an invariant cytotoxic lymphocyte (CTL)-restricted gag epitope required for retroviral structure and replication and implicated in long-term nonprogression to AIDS. The presence of wild-type sequence argues these mutations are involved in immune escape, whereas its reversion to a sequence seen only in the sooty mangabey reservoir suggests an alternate means of controlling infection. Surveillance and molecular characterization of circulating strains are essential for continued development of monitoring tools and may provide greater insight into the reduced pathogenicity of HIV-2.

  9. Identification of Two New HLA-A*1101-Restricted Tax Epitopes Recognized by Cytotoxic T Lymphocytes in an Adult T-Cell Leukemia Patient after Hematopoietic Stem Cell Transplantation

    PubMed Central

    Harashima, Nanae; Tanosaki, Ryuji; Shimizu, Yukiko; Kurihara, Kiyoshi; Masuda, Takao; Okamura, Jun; Kannagi, Mari

    2005-01-01

    We previously reported that Tax-specific CD8+ cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo. PMID:16014972

  10. Identification of two new HLA-A*1101-restricted tax epitopes recognized by cytotoxic T lymphocytes in an adult T-cell leukemia patient after hematopoietic stem cell transplantation.

    PubMed

    Harashima, Nanae; Tanosaki, Ryuji; Shimizu, Yukiko; Kurihara, Kiyoshi; Masuda, Takao; Okamura, Jun; Kannagi, Mari

    2005-08-01

    We previously reported that Tax-specific CD8(+) cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

  11. Definition of the region on NS3 which contains multiple epitopes recognized by dengue virus serotype-cross-reactive and flavivirus-cross-reactive, HLA-DPw2-restricted CD4+ T cell clones.

    PubMed

    Okamoto, Y; Kurane, I; Leporati, A M; Ennis, F A

    1998-04-01

    The epitopes recognized by six CD4+ CD8- cytotoxic T lymphocyte (CTL) clones established from a dengue-3 virus-immune donor were defined. (i) Three CTL clones, JK10, JK34 and JK39, were cross-reactive for dengue virus types 1-4. (ii) One clone, JK28, was cross-reactive for dengue virus types 1-4 and West Nile virus. (iii) Two clones, JK26 and JK49, were cross-reactive for dengue virus types 1-4, West Nile virus and yellow fever virus. The clones, except for JK49, recognized the same epitope on NS3 in an HLA-DPw2-restricted fashion. The smallest synthetic peptide recognized by the five CTL clones was a 10 aa peptide which comprises aa 255-264 on dengue virus NS3. JK49 recognized the overlapping epitope which comprises aa 257-266 in an HLA-DPw2-restricted fashion. Analysis of T cell receptor (TCR) usage by these T cell clones revealed that (i) JK10 and JK34 use V alpha11, and JK34 and JK28 use V beta23, and (ii) the amino acid sequences of the V(D)J junctional region of the TCR were different among these five CTL clones. There were, however, single amino acid conservations among TCRs of some of these T cell clones. These results indicate that the region on NS3 which comprises aa 255-266 contains multiple epitopes recognized by dengue serotype-cross-reactive and flavivirus-cross-reactive CD4+ CTL in an HLA-DPw2-restricted fashion and that a single epitope can be recognized by T cells which have heterogeneous virus specificities.

  12. Identification of an HLA-A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein prostein.

    PubMed

    Kiessling, A; Stevanovic, S; Füssel, S; Weigle, B; Rieger, M A; Temme, A; Rieber, E P; Schmitz, M

    2004-03-08

    The development of T-cell-based immunotherapies of cancer largely depends on the availability of tumour-associated antigens capable of eliciting tumour-directed cytotoxic T-cell responses. In prostate cancer, the number of antigens defined as suitable targets of cytotoxic T lymphocytes (CTLs) is still limited. Recently, prostein was identified as a transmembrane protein that is highly restricted to prostate tissues. In our study, prostein transcripts were found to be abundant in both malignant and nonmalignant prostate tissue samples. To identify immunogenic CD8+ T-cell epitopes, human leucocyte antigen-A(*)0201-binding peptides were selected from the amino-acid sequence of prostein and were used for the in vitro stimulation of CD8+ T lymphocytes. Specific CTLs were raised against the prostein-derived peptide CLAAGITYV that were capable of lysing prostate cancer cells, indicating that this peptide is naturally generated by tumour cells. Our data suggest that prostein is a suitable candidate to be included in a T-cell-based immunotherapy of prostate cancer.

  13. Limited effect on NS3-NS4A protein cleavage after alanine substitutions within the immunodominant HLA-A2-restricted epitope of the hepatitis C virus genotype 3a non-structural 3/4A protease.

    PubMed

    Ahlén, Gustaf; Chen, Antony; Roe, Barbara; Falkeborn, Tina; Frelin, Lars; Hall, William W; Sällberg, Matti; Söderholm, Jonas

    2012-08-01

    It has been well established that immunological escape mutations within the hepatitis C virus genotype (gt) 1a non-structural (NS) 3/4A protease are partly prevented by a reduction in viral protease fitness. Surprisingly little is known about whether similar mutations affect proteases from other genotypes. In the present study, we assessed both the HLA-A2-restricted CTL response and gt3a NS3/4A protease fitness. Similar to gt1, the 1073-1081 epitope was immunodominant within the gt3a-specific HLA-A2-restricted CTL response, despite sequence similarity of only 56 % between the gt1a and gt3a genes. However, unlike the gt1a NS3/4A protease, all residues within the gt3a 1073-1081 epitope could be replaced sequentially by alanine while retaining protease activity, at least in part.

  14. Characterization of cross-reactive CD8+ T-cell recognition of HLA-A2-restricted HIV-Gag (SLYNTVATL) and HCV-NS5b (ALYDVVSKL) epitopes in individuals infected with human immunodeficiency and hepatitis C viruses.

    PubMed

    Vali, Bahareh; Tohn, Robert; Cohen, Michael J; Sakhdari, Ali; Sheth, Prameet M; Yue, Feng Yun; Wong, David; Kovacs, Colin; Kaul, Rupert; Ostrowski, Mario A

    2011-01-01

    The immunologic mechanisms underlying the faster progression of hepatitis C virus (HCV) disease in the presence of human immunodeficiency virus (HIV) coinfection are not clearly understood. T-cell cross-reactivity between HCV and influenza virus-specific epitopes has been associated with rapid progression of HCV disease (S. Urbani, B. Amadei, P. Fisicaro, M. Pilli, G. Missale, A. Bertoletti, and C. Ferrari, J. Exp. Med. 201:675-680, 2005). We asked whether T-cell cross-reactivity between HCV and HIV could exist during HCV/HIV coinfection and affect pathogenesis. Our search for amino acid sequence homology between the HCV and HIV proteomes revealed two similar HLA-A2-restricted epitopes, HIV-Gag (SLYNTVATL [HIV-SL9]) and HCV-NS5b (ALYDVVSKL [HCV-AL9]). We found that 4 out of 20 HLA-A2-positive (HLA-A2(+)) HIV-infected individuals had CD8(+) T cells that recognized both the HIV-SL9 and HCV-AL9 epitopes. However, the AL9 epitope was generally shown to be a weak agonist. Although HCV-monoinfected individuals in our study did not show AL9-specific responses, we found that about half of HCV/HIV-coinfected individuals had dual responses to both epitopes. High dual T-cell recognition among coinfected subjects was usually due to separate T-cell populations targeting each epitope, as determined by pentamer staining. The one individual demonstrating cross-reactive T cells to both epitopes showed the most advanced degree of liver disease. In coinfected individuals, we observed a positive correlation between the magnitudes of T-cell responses to both the SL9 and the AL9 epitopes, which was also positively associated with the clinical parameter of liver damage. Thus, we find that HIV infection induces T cells that can cross-react to heterologous viruses or prime for T cells that are closely related in sequence. However, the induction of cross-reactive T cells may not be associated with control of disease caused by the heterologous virus. This demonstrates that degeneracy of HIV

  15. Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid β peptide

    PubMed Central

    Robert, Remy; Lefranc, Marie-Paule; Ghochikyan, Anahit; Agadjanyan, Michael G.; Cribbs, David H.; Van Nostrand, William E.; Wark, Kim L.; Dolezal, Olan

    2011-01-01

    Over the last decade, the potential of antibodies as therapeutic strategies to treat Alzheimer’s disease (AD) has been growing, based on successful experimental and clinical trials in transgenic mice. Despite, undesirable side effects in humans using an active immunization approach, immunotherapy still remains one of the most promising treatments for AD. In this study, we analyzed the V genes of twelve independently isolated monoclonal antibodies raised against the N-terminal immunodominant epitope of the amyloid β peptide (Aβ or A beta). Surprisingly, we found a high and unusual level of restriction in the VH/VL pairing of these antibodies. Moreover, these antibodies mostly differ in their heavy chain complementary determining region 3 (HCDR3) and the residues in the antibodies which contact Aβ are already present in the germline V-genes. Based on these observations and or co-crystal structures of antibodies with Aβ, the aim of the current study was to better understand the role of antibody V-domains, HCDR3 regions, key contact residue (H58) and germline encoded residues in Aβ recognition. For that purpose, we designed and produced a range of recombinant Fab constructs. All the Fabs were tested and compared by surface plasmon resonance on Aβ1–16, Aβ1–42 high molecular weight and Aβ1–42 low molecular weight soluble oligomers. Although all the Fabs recognized the Aβ1–16 peptide and the Aβ1–42 high molecular weight soluble oligomers, they did not bind the Aβ1–42 low molecular weight soluble oligomers. Furthermore, we demonstrated that: (1) an aromatic residue at position H58 in the antibody is essential in the recognition of Aβ and (2) Fabs based on germline V-genes bind to Aβ monomers with a low affinity. These findings may have important implications in designing more effective therapeutic antibodies against Aβ. PMID:20970857

  16. Antigenicity and predefined specificities of the multi-epitope vaccine in candidate consisting of neutralizing epitope and mutated epitopes suggested a new way against HIV-1 mutation.

    PubMed

    Tian, H; Xiao, Y; Qin, L; Chen, Y H

    2001-12-01

    A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.

  17. Thyroglobulin autoantibodies switch to immunoglobulin (Ig)G1 and IgG3 subclasses and preserve their restricted epitope pattern after 131I treatment for Graves' hyperthyroidism: the activity of autoimmune disease influences subclass distribution but not epitope pattern of autoantibodies.

    PubMed

    Latrofa, F; Ricci, D; Montanelli, L; Piaggi, P; Mazzi, B; Bianchi, F; Brozzi, F; Santini, P; Fiore, E; Marinò, M; Tonacchera, M; Vitti, P

    2014-12-01

    The subclass distribution of thyroglobulin autoantibodies (TgAb) is debated, whereas their epitope pattern is restricted. Radioidine ((131)I) treatment for Graves' disease (GD) induces a rise in TgAb levels, but it is unknown whether it modifies subclass distribution and epitope pattern of TgAb as well. We collected sera from GD patients before (131) I treatment and 3 and 6 months thereafter. We measured total TgAb, TgAb light chains and TgAb subclasses by enzyme-linked immunosorbent assay (ELISA) in 25 patients. We characterized the TgAb epitope pattern in 30 patients by inhibiting their binding to (125-) (I) Tg by a pool of four TgAb-Fab (recognizing Tg epitope regions A, B, C and D) and to Tg in ELISA by each TgAb-Fab. Total TgAb immunoglobulin (Ig)G rose significantly (P = 0.024). TgAb κ chains did not change (P = 0.052), whereas TgAb λ chains increased significantly (P = 0.001) and persistently. We observed a significant rise in IgG1 and IgG3 levels after (131)I (P = 0.008 and P = 0.006, respectively), while IgG2 and IgG4 levels did not change. The rise of IgG1 was persistent, that of IgG3 transient. The levels of inhibition of TgAb binding to Tg by the TgAb-Fab pool were comparable. A slight, non-significant reduction of the inhibition by the immune-dominant TgAb-Fab A was observed 3 and 6 months after (131)I. We conclude that (131)I treatment for GD increases the levels of the complement-activating IgG1 and IgG3 subclasses and does not influence significantly the epitope pattern of TgAb. In autoimmune thyroid disease subclass distribution of autoantibodies is dynamic in spite of a stable epitope pattern.

  18. Thyroglobulin autoantibodies switch to immunoglobulin (Ig)G1 and IgG3 subclasses and preserve their restricted epitope pattern after 131I treatment for Graves' hyperthyroidism: the activity of autoimmune disease influences subclass distribution but not epitope pattern of autoantibodies

    PubMed Central

    Latrofa, F; Ricci, D; Montanelli, L; Piaggi, P; Mazzi, B; Bianchi, F; Brozzi, F; Santini, P; Fiore, E; Marinò, M; Tonacchera, M; Vitti, P

    2014-01-01

    The subclass distribution of thyroglobulin autoantibodies (TgAb) is debated, whereas their epitope pattern is restricted. Radioidine (131I) treatment for Graves' disease (GD) induces a rise in TgAb levels, but it is unknown whether it modifies subclass distribution and epitope pattern of TgAb as well. We collected sera from GD patients before 131I treatment and 3 and 6 months thereafter. We measured total TgAb, TgAb light chains and TgAb subclasses by enzyme-linked immunosorbent assay (ELISA) in 25 patients. We characterized the TgAb epitope pattern in 30 patients by inhibiting their binding to 125-ITg by a pool of four TgAb-Fab (recognizing Tg epitope regions A, B, C and D) and to Tg in ELISA by each TgAb-Fab. Total TgAb immunoglobulin (Ig)G rose significantly (P = 0·024). TgAb κ chains did not change (P = 0·052), whereas TgAb λ chains increased significantly (P = 0·001) and persistently. We observed a significant rise in IgG1 and IgG3 levels after 131I (P = 0·008 and P = 0·006, respectively), while IgG2 and IgG4 levels did not change. The rise of IgG1 was persistent, that of IgG3 transient. The levels of inhibition of TgAb binding to Tg by the TgAb-Fab pool were comparable. A slight, non-significant reduction of the inhibition by the immune-dominant TgAb-Fab A was observed 3 and 6 months after 131I. We conclude that 131I treatment for GD increases the levels of the complement-activating IgG1 and IgG3 subclasses and does not influence significantly the epitope pattern of TgAb. In autoimmune thyroid disease subclass distribution of autoantibodies is dynamic in spite of a stable epitope pattern. PMID:25134846

  19. HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new Plasmodium falciparum epitopes by IFN-gamma ELISPOT.

    PubMed

    González, J M; Peter, K; Esposito, F; Nebié, I; Tiercy, J M; Bonelo, A; Arévalo-Herrera, M; Valmori, D; Romero, P; Herrera, S; Corradin, G; López, J A

    2000-10-01

    The role of antigen specific CD8+ T-lymphocytes in mediating protection against sporozoite-induced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-specific CD8+ T-lymphocytes. Nevertheless, the low frequency of circulating specific cells together with the lack of sensitive methods to quantify them has hampered the direct assessment of their function. Using a combination of short-term cell culture and IFN-gamma ELISPOT, we studied CD8+ T-lymphocyte responses to a panel of HLA-A*0201 binding peptides. In addition to confirming the response to already described epitopes, we also identified five new CD8+ T-lymphocyte epitopes. These epitopes are presented in pre-erythrocytic stages gene products of Plasmodium falciparum 7G8 strain and correspond to the following protein segments: circumsporozoite (CS) 64-72, 104-113, 299-308 and 403-411; liver stage antigen (LSA-1) repeat region; sporozoite surface protein 2 or thrombospondin related anonymous protein (SSP2/TRAP) 78-88 and 504-513. Four of these peptides are conserved amongst all published sequences of P. falciparum strains. We conclude that the modified IFN-gamma ELISPOT assay is a sensitive technique to monitor antigen-specific CD8+ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the efficacy of malaria subunit vaccines.

  20. The life span of major histocompatibility complex-peptide complexes influences the efficiency of presentation and immunogenicity of two class I-restricted cytotoxic T lymphocyte epitopes in the Epstein-Barr virus nuclear antigen 4

    PubMed Central

    1996-01-01

    We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11- positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT- specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide- mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT

  1. Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8(+) T cell responses to HLA class I-restricted wild-type sequence p53 peptides.

    PubMed

    Sakakura, Koichi; Chikamatsu, Kazuaki; Furuya, Nobuhiko; Appella, Ettore; Whiteside, Theresa L; Deleo, Albert B

    2007-10-01

    Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Six HLA-A2 or HLA-A24-restricted wt p53 peptides were evaluated for their ex vivo immunogenicity and their potential for use in cancer vaccines. Peripheral blood mononuclear cells (PBMC) obtained from HLA-A*0201(+) and/or HLA-A*2402(+) normal donors and subjects with squamous cell carcinoma of the head and neck (SCCHN) were analyzed for p53 peptide-specific reactivity in ELISPOT IFN-gamma assays. CD8(+) T cells in 7/10 normal donors (HD) and 11/23 subjects with SCCHN responded to at least one of the wt p53 peptides. CD8(+) T cell precursors responsive to wt p53 epitopes were detected in the circulation of most subjects with early disease, and an elevated blood Tc(1)/Tc(2) ratio distinguished wt p53 peptide responders from non-responders. The identification of multiple wt p53 peptides able to induce cytolytic T lymphocytes in most subjects with cancer promotes the development of multi-epitope p53 vaccines.

  2. Novel immunodominant peptide presentation strategy: a featured HLA-A*2402-restricted cytotoxic T-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein.

    PubMed

    Liu, Jun; Wu, Peng; Gao, Feng; Qi, Jianxun; Kawana-Tachikawa, Ai; Xie, Jing; Vavricka, Christopher J; Iwamoto, Aikichi; Li, Taisheng; Gao, George F

    2010-11-01

    Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β(2)-microglobulin (β(2)m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.

  3. Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

    PubMed Central

    Andersson, Ellen Christina; Hansen, Bjarke Endel; Jacobsen, Helle; Madsen, Lars S.; Andersen, Claus B.; Engberg, Jan; Rothbard, Jonathan B.; McDevitt, Grete Sønderstrup; Malmström, Vivianne; Holmdahl, Rikard; Svejgaard, Arne; Fugger, Lars

    1998-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints. PMID:9636191

  4. In Silico Analysis of Six Known Leishmania major Antigens and In Vitro Evaluation of Specific Epitopes Eliciting HLA-A2 Restricted CD8 T Cell Response

    PubMed Central

    Seyed, Negar; Zahedifard, Farnaz; Safaiyan, Shima; Gholami, Elham; Doustdari, Fatemeh; Azadmanesh, Kayhan; Mirzaei, Maryam; Saeedi Eslami, Nasir; Khadem Sadegh, Akbar; Eslami far, Ali; Sharifi, Iraj; Rafati, Sima

    2011-01-01

    Background As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for

  5. Structure and Function of HLA-A*02-Restricted Hantaan Virus Cytotoxic T-Cell Epitope That Mediates Effective Protective Responses in HLA-A2.1/Kb Transgenic Mice

    PubMed Central

    Ma, Ying; Cheng, Linfeng; Yuan, Bin; Zhang, Yusi; Zhang, Chunmei; Zhang, Yun; Tang, Kang; Zhuang, Ran; Chen, Lihua; Yang, Kun; Zhang, Fanglin; Jin, Boquan

    2016-01-01

    Hantavirus infections cause severe emerging diseases in humans and are associated with high mortality rates; therefore, they have become a global public health concern. Our previous study showed that the CD8+ T-cell epitope aa129–aa137 (FVVPILLKA, FA9) of the Hantaan virus (HTNV) nucleoprotein (NP), restricted by human leukocyte antigen (HLA)-A*02, induced specific CD8+ T-cell responses that controlled HTNV infection in humans. However, the in vivo immunogenicity of peptide FA9 and the effect of FA9-specific CD8+ T-cell immunity remain unclear. Here, based on a detailed structural analysis of the peptide FA9/HLA-A*0201 complex and functional investigations using HLA-A2.1/Kb transgenic (Tg) mice, we found that the overall structure of the peptide FA9/HLA-A*0201 complex displayed a typical MHC class I fold with Val2 and Ala9 as primary anchor residues and Val3 and Leu7 as secondary anchor residues that allow peptide FA9 to bind tightly with an HLA-A*0201 molecule. Residues in the middle portion of peptide FA9 extruding out of the binding groove may be the sites that allow for recognition by T-cell receptors. Immunization with peptide FA9 in HLA-A2.1/Kb Tg mice induced FA9-specific cytotoxic T-cell responses characterized by the induction of high expression levels of interferon-γ, tumor necrosis factor-α, granzyme B, and CD107a. In an HTNV challenge trial, significant reductions in the levels of both the antigens and the HTNV RNA loads were observed in the liver, spleen, and kidneys of Tg mice pre-vaccinated with peptide FA9. Thus, our findings highlight the ability of HTNV epitope-specific CD8+ T-cell immunity to control HTNV and support the possibility that the HTNV-NP FA9 peptide, naturally processed in vivo in an HLA-A*02-restriction manner, may be a good candidate for the development HTNV peptide vaccines. PMID:27551282

  6. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

  7. Development of a T cell receptor targeting an HLA-A*0201 restricted epitope from the cancer-testis antigen SSX2 for adoptive immunotherapy of cancer.

    PubMed

    Abate-Daga, Daniel; Speiser, Daniel E; Chinnasamy, Nachimuthu; Zheng, Zhili; Xu, Hui; Feldman, Steven A; Rosenberg, Steven A; Morgan, Richard A

    2014-01-01

    The clinical success of adoptive immunotherapy of cancer relies on the selection of target antigens that are highly expressed in tumor cells but absent in essential normal tissues. A group of genes that encode the cancer/testis or cancer germline antigens have been proposed as ideal targets for immunotherapy due to their high expression in multiple cancer types and their restricted expression in immunoprivileged normal tissues. In the present work we report the isolation and characterization of human T cell receptors (TCRs) with specificity for synovial sarcoma X breakpoint 2 (SSX2), a cancer/testis antigen expressed in melanoma, prostate cancer, lymphoma, multiple myeloma and pancreatic cancer, among other tumors. We isolated seven HLA-A2 restricted T cell receptors from natural T cell clones derived from tumor-infiltrated lymph nodes of two SSX2-seropositive melanoma patients, and selected four TCRs for cloning into retroviral vectors. Peripheral blood lymphocytes (PBL) transduced with three of four SSX2 TCRs showed SSX241-49 (KASEKIFYV) peptide specific reactivity, tumor cell recognition and tetramer binding. One of these, TCR-5, exhibited tetramer binding in both CD4 and CD8 cells and was selected for further studies. Antigen-specific and HLA-A*0201-restricted interferon-γ release, cell lysis and lymphocyte proliferation was observed following culture of TCR engineered human PBL with relevant tumor cell lines. Codon optimization was found to increase TCR-5 expression in transduced T cells, and this construct has been selected for development of clinical grade viral vector producing cells. The tumor-specific pattern of expression of SSX2, along with the potent and selective activity of TCR-5, makes this TCR an attractive candidate for potential TCR gene therapy to treat multiple cancer histologies.

  8. Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.

    PubMed

    Bounds, Callie E; Hu, Jiafen; Cladel, Nancy M; Balogh, Karla; Christensen, Neil D

    2011-02-01

    The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

  9. Characterization of cloned class I MHC-restricted, CD8+ anti-Meth A cytotoxic T-lymphocytes: recognition of an epitope derived from the Meth A gp110 tumor rejection antigen.

    PubMed

    Fassanito, M A; Loftus, D; De Leo, R M; Law, L W; Appella, E; De Leo, A B

    1994-08-15

    Meth A gp110 has been tentatively identified as a tumor rejection antigen. Following isolation of a class I major histocompatibility complex (MHC)-restricted, CD8+ anti-Meth A cytotoxic T-lymphocyte (CTL), we sought to determine whether the determinant recognized by this CTL was: (a) functional in tumor rejection of Meth A sarcoma; and (b) derived from Meth A gp110. Initially, we isolated an anti-Meth A CTL-resistant variant of Meth A sarcoma, Meth A4R, by immunoselection. The results of the subsequent analysis of Meth A4R cells showed the CTL-defined determinant as having a functional role in transplantation rejection of Meth A sarcoma. Walker et al. (Proc. Natl. Acad. Sci. USA, 89: 7915-7918, 1993) showed that the cationic lipid, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium-methyl sulfate, mediated delivery of a recombinant glycoprotein into the cytosol of target cells, making it available for processing and presentation by class I MHC molecules. As a result, the cells were sensitized for cytolysis by a class I MHC-restricted CD8+ CTL, which recognized an epitope expressed by the glycoprotein. In a similar manner, we treated the SV40-transformed BALB/c cell line, SVBalb, which is relatively insensitive to cytolysis by the anti-Meth A CTL, with Meth A gp110 and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate. The sensitivities of the treated cells and control cell lines to the anti-Meth A CTL were then examined. The results of these experiments permit us to conclude that the determinant recognized by the anti-Meth A CTL line is derived from Meth A gp110.

  10. Fine specificity analysis of an HLA-A2.1-restricted immunodominant T cell epitope derived from human alpha-fetoprotein.

    PubMed

    Meng, W S; Butterfield, L H; Ribas, A; Heller, J B; Dissette, V B; Glaspy, J A; McBride, W H; Economou, J S

    2000-11-01

    Human alpha-fetoprotein (AFP) is a potentially important target for the immunotherapy of hepatocellular carcinoma (HCC). AFP(542-550) (GVALQTMKQ) is one of several HLA-A2.1-restricted immunodominant AFP peptides that consistently generate AFP-specific T cell responses in human T cell cultures and in HLA-A2.1/K(b) transgenic (A2.1 tg) mice. We performed a fine specificity analysis of this nonamer to determine which amino acid side chains were critical for T cell priming and recognition. Using peptide-pulsed dendritic cells (DC) as an immunization strategy, we characterized the effects of AFP(542-550) amino acid substitutions on priming and recognition in A2.1 tg mice. Replacing the glutamine at anchor position 9 with a leucine enhanced MHC binding and AFP-specific T cell responses. Substitution of leucine at non-anchor position 4 with an alanine did not alter binding but greatly diminished T cell recognition. Computer-generated three-dimensional models provided the structural rationale for these observed effects in MHC binding and T cell responses resulted from the modifications in the AFP(542-550) sequence.

  11. The Immune Epitope Database 2.0

    PubMed Central

    Vita, Randi; Zarebski, Laura; Greenbaum, Jason A.; Emami, Hussein; Hoof, Ilka; Salimi, Nima; Damle, Rohini; Sette, Alessandro; Peters, Bjoern

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ∼99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009. PMID:19906713

  12. Enhancement of antigen acquisition by dendritic cells and MHC class II-restricted epitope presentation to CD4+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection.

    PubMed

    Mwangi, Waithaka; Brown, Wendy C; Splitter, Gary A; Zhuang, Yan; Kegerreis, Kimberly; Palmer, Guy H

    2005-08-01

    Induction of immune responses against microbial antigens using DNA is an attractive strategy to mimic the immunity induced by live vaccines. Although DNA vaccines are efficacious in murine models, the requirement for multiple immunizations using high doses in outbred animals and humans has hindered deployment. This requirement is, in part, a result of poor vaccine spreading and suboptimal DC transfection efficiency. Incorporation of a signal that directs intercellular spreading of a DNA-encoded antigen is proposed to mimic live vaccine spreading and increase dendritic cell (DC) presentation. Bovine herpes virus 1 tegument protein, BVP22, is capable of trafficking to surrounding cells. To test the hypothesis that BVP22 enhances spreading and antigen presentation to CD4+ T cells, a DNA construct containing BVP22, fused in-frame to a sequence encoding a T cell epitope of Anaplasma marginale, was generated. A construct with reversed BVP22 sequence served as a negative control. Immunocytometric analysis of transfected primary keratinocytes, human embryonic kidney 293, COS-7, and Chinese hamster ovary cells showed that BVP22 enhanced intercellular spreading by > or = 150-fold. Flow cytometric analysis of antigen-presenting cells (APCs) positively selected from cocultures of transfected cells and APCs showed that 5% of test APCs were antigen-positive, compared with 0.6% of control APCs. Antigen-specific CD4+ T cell proliferation demonstrated that BVP22 enhanced DC antigen presentation by > or = 20-fold. This first report of the ability of BVP22 to increase DNA-encoded antigen acquisition by DCs and macrophages, with subsequent enhancement of major histocompatibility complex class II-restricted CD4+ T cell responses, supports incorporating a spreading motif in a DNA vaccine to target CD4+ T cell-dependent immunity in outbred animals.

  13. HLA-A02:01-Restricted Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP11/12 Preferentially Recall Polyfunctional Effector Memory CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect “Humanized” HLA-A*02:01 Transgenic Mice Against Ocular Herpes

    PubMed Central

    Srivastava, Ruchi; Khan, Arif A.; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P.; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T.; Huang, Jiawei; Scarfone, Vanessa M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2014-01-01

    The Herpes Simplex Virus type 1 virion tegument phosphoprotein 11/12 (HSV-1 VP11/12) is a major antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether and which VP11/12-epitope-specific CD8+ T cells play a role in the “natural” protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 716 amino acids sequence of VP11/12. Three out of ten epitopes exhibited high to moderate binding affinity to HLA-A*02:01 molecules. In ten sequentially studied HLA-A*02:01 positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust and polyfunctional effector CD8+ T-cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266–74, VP11/12220–228 and VP11/12702–710. Interestingly, ASYMP individuals had significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory T cells (TEM) specific to the three epitopes, compared to symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of an effective T-cell-based herpes vaccine. PMID:25617474

  14. Design of multi-epitope, analogue-based cancer vaccines.

    PubMed

    Fikes, John D; Sette, Alessandro

    2003-09-01

    The current objective of our cancer programme is to develop an effective vaccine based on rationally designed T cell epitope analogues, for use in the adjuvant setting for non-small cell lung cancer (NSCLC) and colon cancer. Analogue epitopes, enhanced for either human leukocyte antigen (HLA) binding or T cell receptor (TCR) signalling, have been shown to be more effective at breaking immunological tolerance than cognate wild-type epitopes. Although encouraging early-phase clinical data has been obtained by others using a limited number of HLA-A2-restricted epitope analogues, the clinical benefits and immune correlates for vaccines comprised of multiple epitope analogues restricted by multiple HLA supertypes remains to be investigated. Clinical studies are currently being conducted on EP-2101, a prototype vaccine that delivers multiple HLA-A2-restricted analogue epitopes. In parallel, fixed anchor and heteroclitic analogues restricted by three other commonly expressed HLA supertypes are being identified. These analogues will be incorporated into future vaccines including optimised minigenes (epigenes) and tested in preclinical and clinical studies addressing various different cancer indications.

  15. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

    Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on

  16. Targeting Non-classical Myelin Epitopes to Treat Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Wang, Xiaohua; Zhang, Jintao; Baylink, David J.; Li, Chih-Huang; Watts, Douglas M.; Xu, Yi; Qin, Xuezhong; Walter, Michael H.; Tang, Xiaolei

    2016-01-01

    Qa-1 epitopes, the peptides that bind to non-classical major histocompatibility complex Ib Qa-1 molecules and are recognized by Qa-1-restricted CD8+ regulatory T (Treg) cells, have been identified in pathogenic autoimmune cells that attack myelin sheath in experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis [MS]). Additionally, immunization with such epitopes ameliorates the EAE. However, identification of such epitopes requires knowledge of the pathogenic autoimmune cells which are largely unknown in MS patients. Hence, we asked whether the CD8+ Treg cells could directly target the myelin sheath to ameliorate EAE. To address this question, we analyzed Qa-1 epitopes in myelin oligodendrocyte glycoprotein (MOG that is a protein in myelin sheath). Here, we report identification of a MOG-specific Qa-1 epitope. Immunization with this epitope suppressed ongoing EAE, which was abrogated by CD8+ T cell depletion. Additionally, the epitope immunization activated the epitope-specific CD8+ T cells which specifically accumulated in the CNS-draining cervical lymph nodes. Finally, CD8+ T cells primed by the epitope immunization transferred EAE suppression. Hence, this study reveals a novel regulatory mechanism mediated by the CD8+ Treg cells. We propose that immunization with myelin-specific HLA-E epitopes (human homologues of Qa-1 epitopes) is a promising therapy for MS. PMID:27796368

  17. Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

    PubMed

    Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep

    2015-12-01

    Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

  18. Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

    PubMed

    Novak, E J; Liu, A W; Gebe, J A; Falk, B A; Nepom, G T; Koelle, D M; Kwok, W W

    2001-06-01

    T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

  19. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    PubMed

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

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

    PubMed Central

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

    2017-01-01

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

  1. Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation

    PubMed Central

    Schellens, Ingrid M.; Meiring, Hugo D.; Hoof, Ilka; Spijkers, Sanne N.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Costa, Ana I.; Vennema, Harry; Keşmir, Can; van Baarle, Debbie; van Els, Cécile A. C. M.

    2015-01-01

    Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. ­Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV. PMID:26579122

  2. In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies.

    PubMed

    Tang, Xu-Dong; Guo, Shu-Liang; Wang, Guo-Zhen; Li, Ning; Wu, Yu-Yun; Fang, Dian-Chun; Fan, Ya-Han; Yang, Shi-Ming

    2014-01-01

    Previous studies have indicated that heparanase (Hpa) might represent a candidate universal tumor-associated antigen. However, vaccine therapy targeting only one cytotoxic T lymphocyte (CTL) epitope is suboptimal in preventing cancer. In the present study, we designed heparanase multi-epitope vaccines to increase the immune response to standard single heparanase epitopes. The results showed that multi-epitope vaccines Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 induced higher Hpa-specific lysis of various cancer cells from different tissues in a HLA-A2-restricted and heparanase-specific manner compared with the single epitope vaccines Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363, both in vitro and ex vivo. Heparanase multi-epitope vaccines not only induced the heparanase-specific CTL to lyse tumor cells but also increased CTL secretion of interferon-γ. However, these heparanase-specific CTL did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirms the safety of these multi-epitope vaccines. Therefore, the present study provides theoretical evidence for the use of heparanase multi-epitope vaccines for clinical application.

  3. Th1 epitope selection for clinically effective cancer vaccines.

    PubMed

    Disis, Mary L; Watt, William C; Cecil, Denise L

    2014-10-01

    New cancer immunotherapies mark progress in our understanding of tumor biology and harnessing the immune system's management of self. However, protein- and peptide-based vaccines are not yet consistently efficacious. Recent work uncovers principles governing the genesis of T helper type-restrictive immunity to self-antigens elicited by vaccine epitopes, enabling vaccines to skew the balance from tolerogenic Type II (Th2) to inflammatory Type I (Th1) T cells, and invigorating this cancer immunotherapeutic approach.

  4. Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

    PubMed Central

    Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin

    2015-01-01

    The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated

  5. Epitope specific T-cell responses against influenza A in a healthy population.

    PubMed

    Savic, Miloje; Dembinski, Jennifer L; Kim, Yohan; Tunheim, Gro; Cox, Rebecca J; Oftung, Fredrik; Peters, Bjoern; Mjaaland, Siri

    2016-02-01

    Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.

  6. Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine.

    PubMed

    Ohtake, Junya; Ohkuri, Takayuki; Togashi, Yuji; Kitamura, Hidemitsu; Okuno, Kiyotaka; Nishimura, Takashi

    2014-09-01

    We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4(+) Th1 cells and CD8(+) Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.

  7. Novel and promiscuous CTL epitopes in conserved regions of Gag targeted by individuals with early subtype C HIV type 1 infection from southern Africa.

    PubMed

    Masemola, Agatha M; Mashishi, Tumelo N; Khoury, Greg; Bredell, Helba; Paximadis, Maria; Mathebula, Tiyani; Barkhan, Debra; Puren, Adrian; Vardas, Efthyia; Colvin, Mark; Zijenah, Lynn; Katzenstein, David; Musonda, Rosemary; Allen, Susan; Kumwenda, Newton; Taha, Taha; Gray, Glenda; McIntyre, James; Karim, Salim Abdool; Sheppard, Haynes W; Gray, Clive M

    2004-10-01

    Characterization of optimal CTL epitopes in Gag can provide crucial information for evaluation of candidate vaccines in populations at the epicenter of the HIV-1 epidemic. We screened 38 individuals with recent subtype C HIV-1 infection using overlapping consensus C Gag peptides and hypothesized that unique HLA-restricting alleles in the southern African population would determine novel epitope identity. Seventy-four percent of individuals recognized at least one Gag peptide pool. Ten epitopic regions were identified across p17, p24, and p2p7p1p6, and greater than two-thirds of targeted regions were directed at: TGTEELRSLYNTVATLY (p17, 35%); GPKEPFRDYVDRFFKTLRAEQATQDV (p24, 19%); and RGGKLDKWEKIRLRPGGKKHYMLKHL (p17, 15%). After alignment of these epitopic regions with consensus M and a consensus subtype C sequence from the cohort, it was evident that the regions targeted were highly conserved. Fine epitope mapping revealed that five of nine identified optimal Gag epitopes were novel: HLVWASREL, LVWASRELERF, LYNTVATLY, PFRDYVDRFF, and TLRAEQATQD, and were restricted by unique HLA-Cw*08, HLA-A*30/B*57, HLA-A*29/B*44, and HLA-Cw*03 alleles, respectively. Notably, three of the mapped epitopes were restricted by more than one HLA allele. Although these epitopes were novel and restricted by unique HLA, they overlapped or were embedded within previously described CTL epitopes from subtype B HIV-1 infection. These data emphasize the promiscuous nature of epitope binding and support our hypothesis that HLA diversity between populations can shape fine epitope identity, but may not represent a constraint for universal recognition of Gag in highly conserved domains.

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

    PubMed

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

    2008-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. Results Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite

  11. An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction

    PubMed Central

    Potocnakova, Lenka

    2016-01-01

    Identification of B-cell epitopes is a fundamental step for development of epitope-based vaccines, therapeutic antibodies, and diagnostic tools. Epitope-based antibodies are currently the most promising class of biopharmaceuticals. In the last decade, in-depth in silico analysis and categorization of the experimentally identified epitopes stimulated development of algorithms for epitope prediction. Recently, various in silico tools are employed in attempts to predict B-cell epitopes based on sequence and/or structural data. The main objective of epitope identification is to replace an antigen in the immunization, antibody production, and serodiagnosis. The accurate identification of B-cell epitopes still presents major challenges for immunologists. Advances in B-cell epitope mapping and computational prediction have yielded molecular insights into the process of biorecognition and formation of antigen-antibody complex, which may help to localize B-cell epitopes more precisely. In this paper, we have comprehensively reviewed state-of-the-art experimental methods for B-cell epitope identification, existing databases for epitopes, and novel in silico resources and prediction tools available online. We have also elaborated new trends in the antibody-based epitope prediction. The aim of this review is to assist researchers in identification of B-cell epitopes. PMID:28127568

  12. An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction.

    PubMed

    Potocnakova, Lenka; Bhide, Mangesh; Pulzova, Lucia Borszekova

    2016-01-01

    Identification of B-cell epitopes is a fundamental step for development of epitope-based vaccines, therapeutic antibodies, and diagnostic tools. Epitope-based antibodies are currently the most promising class of biopharmaceuticals. In the last decade, in-depth in silico analysis and categorization of the experimentally identified epitopes stimulated development of algorithms for epitope prediction. Recently, various in silico tools are employed in attempts to predict B-cell epitopes based on sequence and/or structural data. The main objective of epitope identification is to replace an antigen in the immunization, antibody production, and serodiagnosis. The accurate identification of B-cell epitopes still presents major challenges for immunologists. Advances in B-cell epitope mapping and computational prediction have yielded molecular insights into the process of biorecognition and formation of antigen-antibody complex, which may help to localize B-cell epitopes more precisely. In this paper, we have comprehensively reviewed state-of-the-art experimental methods for B-cell epitope identification, existing databases for epitopes, and novel in silico resources and prediction tools available online. We have also elaborated new trends in the antibody-based epitope prediction. The aim of this review is to assist researchers in identification of B-cell epitopes.

  13. Selection of Conserved Epitopes from Hepatitis C Virus for Pan-Populational Stimulation of T-Cell Responses

    PubMed Central

    Molero-Abraham, Magdalena; Lafuente, Esther M.; Flower, Darren R.; Reche, Pedro A.

    2013-01-01

    The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server. PMID:24348677

  14. Restrictive cardiomyopathy

    MedlinePlus

    Cardiomyopathy - restrictive; Infiltrative cardiomyopathy; Idiopathic myocardial fibrosis ... In a case of restrictive cardiomyopathy, the heart muscle is of normal size or slightly enlarged. Most of the time, it also pumps normally. However, it does ...

  15. Design of immunogenic and effective multi-epitope DNA vaccines for melanoma.

    PubMed

    Cho, Hyun-Il; Celis, Esteban

    2012-03-01

    Plasmid DNA vaccination is an attractive way to elicit T cell responses against infectious agents and tumor cells. DNA constructs can be designed to contain multiple T cell epitopes to generate a diverse immune response to incorporate numerous antigens and to reduce limitations due to MHC restriction into a single entity. We have prepared cDNA plasmid constructs containing several mouse T cell epitopes connected by either furin-sensitive or furin-resistant linkers and studied the effects of a cationic cell-penetrating sequence from HIV-tat. Significant CD8 T cell responses were obtained with multi-epitope DNA vaccines followed by in vivo electroporation regardless of the type of linker used and whether the construct had the HIV-tat sequence. The magnitude of immune responses was very similar to all CD8 T cell epitopes contained within each vaccine construct, indicating the absence of immunodominance. Incorporating a T helper epitope into the constructs increased the T cell responses. Prophylactic and therapeutic antitumor responses against B16 melanoma were obtained using a construct containing epitopes from melanosomal proteins, indicating that this vaccination was successful in generating responses to self-antigens that potentially may be subjected to immune tolerance. These findings are useful for designing DNA vaccines for a multitude of diseases where T lymphocytes play a protective or therapeutic role.

  16. Automatic Generation of Validated Specific Epitope Sets.

    PubMed

    Carrasco Pro, Sebastian; Sidney, John; Paul, Sinu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro

    2015-01-01

    Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

  17. Predicting population coverage of T-cell epitope-based diagnostics and vaccines

    PubMed Central

    Bui, Huynh-Hoa; Sidney, John; Dinh, Kenny; Southwood, Scott; Newman, Mark J; Sette, Alessandro

    2006-01-01

    Background T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. Results To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at . Conclusion We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups. PMID:16545123

  18. Ligand-induced Epitope Masking

    PubMed Central

    Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

    2016-01-01

    We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

  19. CD8 epitope escape and reversion in acute HCV infection.

    PubMed

    Timm, Joerg; Lauer, Georg M; Kavanagh, Daniel G; Sheridan, Isabelle; Kim, Arthur Y; Lucas, Michaela; Pillay, Thillagavathie; Ouchi, Kei; Reyor, Laura L; Schulze zur Wiesch, Julian; Gandhi, Rajesh T; Chung, Raymond T; Bhardwaj, Nina; Klenerman, Paul; Walker, Bruce D; Allen, Todd M

    2004-12-20

    In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

  20. Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

    PubMed

    Oldstone, M B; Lewicki, H; Homann, D; Nguyen, C; Julien, S; Gairin, J E

    2001-07-01

    Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

  1. An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform

    PubMed Central

    Fridman, Arthur; Finnefrock, Adam C.; Peruzzi, Daniela; Pak, Irene; La Monica, Nicola; Bagchi, Ansuman; Casimiro, Danilo R.; Ciliberto, Gennaro; Aurisicchio, Luigi

    2012-01-01

    Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopiaTM Epitope Discovery SystemTM, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens. PMID:23243589

  2. First report on the antibody verification of MICA epitopes recorded in the HLA epitope registry.

    PubMed

    Duquesnoy, R J; Mostecki, J; Marrari, M; da Silva, A S; da Mata Sousa, L C D; do Monte, S J H

    2014-10-01

    The International Registry of HLA Epitopes (http://epregistry.com.br) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three-dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty-one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

  3. Chimeric virus-like particles for the delivery of an inserted conserved influenza A-specific CTL epitope.

    PubMed

    Cheong, Wan-Shoo; Reiseger, Jessica; Turner, Stephen John; Boyd, Richard; Netter, Hans-Jürgen

    2009-02-01

    The small hepatitis B virus surface antigens (HBsAg-S) have the ability to self-assemble with host-derived lipids into empty non-infectious virus-like particles (VLPs). HBsAg-S VLPs are the sole component of the licensed hepatitis B vaccine, and they are a useful delivery platform for foreign epitopes. To develop VLPs capable of transporting foreign cytotoxic T lymphocyte (CTL) epitopes, HBsAg-S specific CTL epitopes at various sites were substituted with a conserved CTL epitope derived from the influenza matrix protein. Depending on the insertion site, the introduction of the MHC class I A2.1-restricted influenza epitope was compatible with the secretion competence of HBsAg-S indicating that chimeric VLPs were assembled. Immunizations of transgenic HHDII mice with chimeric VLPs induced anti-influenza CTL responses proving that the inserted foreign epitope can be correctly processed and cross-presented. Chimeric VLPs in the absence of adjuvant were able to induce memory T cell responses, which could be recalled by influenza virus infections in the mouse model system. The ability of chimeric HBsAg-S VLPs to induce anti-foreign CTL responses and also with the proven ability to induce humoral immune responses constitute a highly versatile platform for the delivery of selected multiple epitopes to target disease associated infectious agents.

  4. HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T-cell epitope

    PubMed Central

    Nitschke, Katja; Barriga, Alejandro; Schmidt, Julia; Timm, Jörg; Viazov, Sergei; Kuntzen, Thomas; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Gaudieri, Silvana; Rauch, Andri; Lange, Christian M.; Sarrazin, Christoph; Eiermann, Thomas; Sidney, John; Sette, Alessandro; Thimme, Robert; López, Daniel; Neumann-Haefelin, Christoph

    2013-01-01

    Background & Aims HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T-cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Methods Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T-cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. Results The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. Conclusions The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. PMID:23978718

  5. Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.

    PubMed

    Georgiev, O; Bourquin, J P; Gstaiger, M; Knoepfel, L; Schaffner, W; Hovens, C

    1996-02-12

    Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.

  6. Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation

    PubMed Central

    Goulder, P.J.R.; Sewell, A.K.; Lalloo, D.G.; Price, D.A.; Whelan, J.A.; Evans, J.; Taylor, G.P.; Luzzi, G.; Giangrande, P.; Phillips, R.E.; McMichael, A.J.

    1997-01-01

    Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response. PMID:9126923

  7. Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1.

    PubMed

    Li, Hua; Liu, Zu-Qiang; Ding, Jian; Chen, Ying-Hua

    2002-11-01

    Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.

  8. Malondialdehyde epitopes as mediators of sterile inflammation.

    PubMed

    Busch, Clara J; Binder, Christoph J

    2017-04-01

    Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

  9. Recovery of known T-cell epitopes by computational scanning of a viral genome

    NASA Astrophysics Data System (ADS)

    Logean, Antoine; Rognan, Didier

    2002-04-01

    A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list. The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.

  10. Immunoinformatics Approach in Designing Epitope-based Vaccine Against Meningitis-inducing Bacteria (Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae Type b)

    PubMed Central

    Zahroh, Hilyatuz; Ma’rup, Ahmad; Tambunan, Usman Sumo Friend; Parikesit, Arli Aditya

    2016-01-01

    Meningitis infection is one of the major threats during Hajj season in Mecca. Meningitis vaccines are available, but their uses are limited in some countries due to religious reasons. Furthermore, they only give protection to certain serogroups, not to all types of meningitis-inducing bacteria. Recently, research on epitope-based vaccines has been developed intensively. Such vaccines have potential advantages over conventional vaccines in that they are safer to use and well responded to the antibody. In this study, we developed epitope-based vaccine candidates against various meningitis-inducing bacteria, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b. The epitopes were selected from their protein of polysaccharide capsule. B-cell epitopes were predicted by using BCPred, while T-cell epitope for major histocompatibility complex (MHC) class I was predicted using PAProC, TAPPred, and Immune Epitope Database. Immune Epitope Database was also used to predict T-cell epitope for MHC class II. Population coverage and molecular docking simulation were predicted against previously generated epitope vaccine candidates. The best candidates for MHC class I- and class II-restricted T-cell epitopes were MQYGDKTTF, MKEQNTLEI, ECTEGEPDY, DLSIVVPIY, YPMAMMWRNASNRAI, TLQMTLLGIVPNLNK, ETSLHHIPGISNYFI, and SLLYILEKNAEMEFD, which showed 80% population coverage. The complexes of class I T-cell epitopes–HLA-C*03:03 and class II T-cell epitopes–HLA-DRB1*11:01 showed better affinity than standards as evaluated from their ΔGbinding value and the binding interaction between epitopes and HLA molecules. These peptide constructs may further be undergone in vitro and in vivo testings for the development of targeted vaccine against meningitis infection. PMID:27812281

  11. The two-faced T cell epitope

    PubMed Central

    Moise, Leonard; Gutierrez, Andres H.; Bailey-Kellogg, Chris; Terry, Frances; Leng, Qibin; Abdel Hady, Karim M.; VerBerkmoes, Nathan C.; Sztein, Marcelo B.; Losikoff, Phyllis T.; Martin, William D.; Rothman, Alan L; De Groot, Anne S.

    2013-01-01

    Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally–driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell

  12. 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

  13. Restrictive cardiomyopathies.

    PubMed

    Nihoyannopoulos, Petros; Dawson, David

    2009-12-01

    Restrictive cardiomyopathies constitute a heterogenous group of heart muscle conditions that all have, in common, the symptoms of heart failure. Diastolic dysfunction with preserved systolic function is often the only echocardiographic abnormality that may be noted, although systolic dysfunction may also be an integral part of some specific pathologies, particularly in the most advanced cases such as amyloid infiltration of the heart. By far, the majority of restrictive cardiomyopathies are secondary to a systemic disorder such as amyloidosis, sarcoidosis, scleroderma, haemochromatosis, eosinophilic heart disease, or as a result of radiation treatment. The much more rare diagnosis of idiopathic restrictive cardiomyopathy is supported only by the absence of specific pathology on either endomyocardial biopsies or at post-mortem. Restrictive cardiomyopathy is diagnosed based on medical history, physical examination, and tests: such as blood tests, electrocardiogram, chest X-ray, echocardiography, and magnetic resonance imaging. With its wide availability, echocardiography is probably the most important investigation to identify the left ventricular dysfunction and should be performed early and by groups that are familiar with the wide variety of aetiologies. Finally, on rare occasions, the differential diagnosis from constrictive pericarditis may be necessary.

  14. Intramolecular epitope spreading in Heymann nephritis.

    PubMed

    Shah, Pallavi; Tramontano, Alfonso; Makker, Sudesh P

    2007-12-01

    Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

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

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    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 its 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.

  16. Human monoclonal antibodies that recognize conserved epitopes in the core-lipid A region of lipopolysaccharides.

    PubMed Central

    Pollack, M; Raubitschek, A A; Larrick, J W

    1987-01-01

    Epstein-Barr virus (EBV)-transformed human B lymphocytes were fused with a murine-human heteromyeloma to produce stable hybrid cell lines that secreted human monoclonal antibodies (mAbs) of the IgM class that recognized conserved epitopes in the core-lipid A region of lipopolysaccharides (LPS). Three of the mAbs reacted with epitopes on the lipid A moiety, while a fourth recognized a determinant in the core oligosaccharide. The lipid A-specific mAbs cross-reacted with heterologous rough LPS and with lipid As released by acid hydrolysis of different intact (smooth) LPS. Carbohydrate groups in the O-side chain and core oligosaccharide of isolated, smooth LPS restricted antibody access to antigenic sites on lipid A. Yet, one lipid A-reactive mAb recognized its epitope on the surfaces of a variety of intact bacteria. These findings confirm the presence of highly conserved epitopes in the core-lipid A complex and prove the existence of human B cell clones with the potential for secreting high avidity IgM antibodies that react with these widely shared determinants. Such human mAbs might provide protective activity against disease caused by diverse gram-negative bacteria. Images PMID:2437155

  17. Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells.

    PubMed

    Mishra, Sasmita; Losikoff, Phyllis T; Self, Alyssa A; Terry, Frances; Ardito, Matthew T; Tassone, Ryan; Martin, William D; De Groot, Anne S; Gregory, Stephen H

    2014-05-30

    Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8(+)) and class II-restricted (CD4(+)) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.

  18. Processing and cross-presentation of individual HLA-A, -B, or -C epitopes from NY-ESO-1 or an HLA-A epitope for Melan-A differ according to the mode of antigen delivery.

    PubMed

    Robson, Neil C; McAlpine, Tristan; Knights, Ashley J; Schnurr, Max; Shin, Amanda; Chen, Weisan; Maraskovsky, Eugene; Cebon, Jonathan

    2010-07-15

    The ability of dendritic cells (DCs) to cross-present protein tumor antigens to cytotoxic T lymphocytes (CTLs) underpins the success of therapeutic cancer vaccines. We studied cross-presentation of the cancer/testis antigen, NY-ESO-1, and the melanoma differentiation antigen, Melan-A by human DC subsets. Monocyte-derived DCs (MoDCs) efficiently cross-presented human leukocyte associated (HLA)-A2-restricted epitopes from either a formulated NY-ESO-1/ISCOMATRIX vaccine or when either antigen was mixed with ISCOMATRIX adjuvant. HLA-A2 epitope generation required endosomal acidification and was proteasome-independent for NY-ESO-1 and proteasome-dependent for Melan-A. Both MoDCs and CD1c(+) blood DCs cross-presented NY-ESO-1-specific HLA-A2(157-165)-, HLA-B7(60-72)-, and HLA-Cw3(92-100)-restricted epitopes when formulated as an NY-ESO-1/ISCOMATRIX vaccine, but this was limited when NY-ESO-1 and ISCOMATRIX adjuvant were added separately to the DC cultures. Finally, cross-presentation of NY-ESO-1(157-165)/HLA-A2, NY-ESO-1(60-72)/HLA-B7, and NY-ESO-1(92-100)/HLA-Cw3 epitopes was proteasome-dependent when formulated as immune complexes (ICs) but only proteasome-dependent for NY-ESO-1(60-72)/HLA-B7-restricted cross-presentation facilitated by ISCOMATRIX adjuvant. We demonstrate, for the first time, proteasome-dependent and independent cross-presentation of HLA-A-, B-, and C-restricted epitopes within the same full-length tumor antigen by human DCs. Our findings identify important differences in the capacities of human DC subsets to cross-present clinically relevant, full-length tumor antigens and how vaccine formulation impacts CTL responses in vivo.

  19. The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design

    PubMed Central

    Fleri, Ward; Paul, Sinu; Dhanda, Sandeep Kumar; Mahajan, Swapnil; Xu, Xiaojun; Peters, Bjoern; Sette, Alessandro

    2017-01-01

    The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources. PMID:28352270

  20. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  1. The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design.

    PubMed

    Fleri, Ward; Paul, Sinu; Dhanda, Sandeep Kumar; Mahajan, Swapnil; Xu, Xiaojun; Peters, Bjoern; Sette, Alessandro

    2017-01-01

    The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources.

  2. Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8(+) and CD4(+) T-cell responses with multiple specificities including a novel DR7-restricted epitope.

    PubMed

    Baumgaertner, P; Costa Nunes, C; Cachot, A; Maby-El Hajjami, H; Cagnon, L; Braun, M; Derré, L; Rivals, J-P; Rimoldi, D; Gnjatic, S; Abed Maillard, S; Marcos Mondéjar, P; Protti, M P; Romano, E; Michielin, O; Romero, P; Speiser, D E; Jandus, C

    2016-01-01

    Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8(+) and CD4(+) T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8(+) and NY-ESO-183-99 for CD4(+) T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7(+) patients generated strong CD4(+) T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8(+) and CD4(+) T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

  3. Molecular basis of cross-reactivity among allergen-specific human T cells: T-cell receptor V alpha gene usage and epitope structure.

    PubMed Central

    Mohapatra, S S; Mohapatra, S; Yang, M; Ansari, A A; Parronchi, P; Maggi, E; Romagnani, S

    1994-01-01

    Cross-reactivities between the major grass pollen allergens, at the level of T-cell recognition was examined employing several Lolium perenne I (Lol p I)-specific human T-cell clones. Nine of these Lol p I-specific T-cell clones exhibited cross-recognition of the recombinant Poa pratensis IX (Poa p IX) allergen, rKBG7.2, indicating that these two major antigens of a grass pollen share T-cell epitopes. Furthermore, proliferative responses of two other T-cell clones demonstrated that individual allergens of diverse grass pollens also possess common T-cell epitopes. Examination of the T-cell receptor (TcR) V alpha genes of these T-cell clones indicated that these cloned cells utilized distinct J alpha genes and that nine out of 10 clones possessed V alpha 13 gene. Furthermore, sequence comparisons of several allergenic molecules indicated that this cross-reactivity may be due to the presence of epitope(s) with structure(s) similar to the major T-cell epitope of Poa p IX allergens. Taken together, these results suggest for the first time that the major grass pollen allergens share cross-reacting T-cell epitope(s), and that this cross-reactivity is due to the structural homologies among allergens and restricted usage of TcR V alpha genes. PMID:7510663

  4. The Relationship between B-cell Epitope and Mimotope Sequences.

    PubMed

    Zhang, Chunhua; Li, Yunyun; Tang, Weina; Zhou, Zhiguo; Sun, Pingping; Ma, Zhiqiang

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint.

  5. Modules for C-terminal epitope tagging of Tetrahymena genes

    PubMed Central

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  6. Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors.

    PubMed

    Purbhoo, Marco A; Sutton, Deborah H; Brewer, Joanna E; Mullings, Rebecca E; Hill, Maxine E; Mahon, Tara M; Karbach, Julia; Jäger, Elke; Cameron, Brian J; Lissin, Nikolai; Vyas, Paresh; Chen, Ji-Li; Cerundolo, Vincenzo; Jakobsen, Bent K

    2006-06-15

    Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.

  7. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

    PubMed

    Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

    2012-01-01

    We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

  8. 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

  9. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

    PubMed Central

    Meinl, E; Weber, F; Drexler, K; Morelle, C; Ott, M; Saruhan-Direskeneli, G; Goebels, N; Ertl, B; Jechart, G; Giegerich, G

    1993-01-01

    The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an

  10. Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

    PubMed Central

    Dominguez, Mariana R.; Silveira, Eduardo L. V.; de Vasconcelos, José Ronnie C.; de Alencar, Bruna C. G.; Machado, Alexandre V.; Bruna-Romero, Oscar; Gazzinelli, Ricardo T.; Rodrigues, Mauricio M.

    2011-01-01

    During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development. PMID:21779365

  11. Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

    PubMed Central

    1996-01-01

    An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

  12. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses.

    PubMed

    Smith, Heath A; Rekoske, Brian T; McNeel, Douglas G

    2014-03-26

    Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

  13. Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8(+) T cells.

    PubMed

    Wen, Jinsheng; Tang, William Weihao; Sheets, Nicholas; Ellison, Julia; Sette, Alessandro; Kim, Kenneth; Shresta, Sujan

    2017-03-13

    CD8(+) T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/β receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-γ-ELISPOT and IFN-γ-ICS on CD8(+) T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8(+) T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8(+) T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8(+) T cell responses that reduced infectious ZIKV levels, and CD8(+) T cell depletions confirmed that CD8(+) T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8(+) T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8(+) T cell responses of human relevance.

  14. Viral Evolution and Cytotoxic T Cell Restricted Selection in Acute Infant HIV-1 Infection

    PubMed Central

    Garcia-Knight, Miguel A.; Slyker, Jennifer; Payne, Barbara Lohman; Pond, Sergei L. Kosakovsky; de Silva, Thushan I.; Chohan, Bhavna; Khasimwa, Brian; Mbori-Ngacha, Dorothy; John-Stewart, Grace; Rowland-Jones, Sarah L.; Esbjörnsson, Joakim

    2016-01-01

    Antiretroviral therapy-naive HIV-1 infected infants experience poor viral containment and rapid disease progression compared to adults. Viral factors (e.g. transmitted cytotoxic T- lymphocyte (CTL) escape mutations) or infant factors (e.g. reduced CTL functional capacity) may explain this observation. We assessed CTL functionality by analysing selection in CTL-targeted HIV-1 epitopes following perinatal infection. HIV-1 gag, pol and nef sequences were generated from a historical repository of longitudinal specimens from 19 vertically infected infants. Evolutionary rate and selection were estimated for each gene and in CTL-restricted and non-restricted epitopes. Evolutionary rate was higher in nef and gag vs. pol, and lower in infants with non-severe immunosuppression vs. severe immunosuppression across gag and nef. Selection pressure was stronger in infants with non-severe immunosuppression vs. severe immunosuppression across gag. The analysis also showed that infants with non-severe immunosuppression had stronger selection in CTL-restricted vs. non-restricted epitopes in gag and nef. Evidence of stronger CTL selection was absent in infants with severe immunosuppression. These data indicate that infant CTLs can exert selection pressure on gag and nef epitopes in early infection and that stronger selection across CTL epitopes is associated with favourable clinical outcomes. These results have implications for the development of paediatric HIV-1 vaccines. PMID:27403940

  15. TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys

    PubMed Central

    Osuna, Christa E.; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S. Munir; Letvin, Norman L.

    2014-01-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design. PMID:24743648

  16. TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

    PubMed

    Osuna, Christa E; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S Munir; Letvin, Norman L

    2014-04-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

  17. Epitope Mapping with Random Phage Display Library

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.

    2017-01-01

    Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results. PMID:24515483

  18. Relevant B Cell Epitopes in Allergic Disease

    PubMed Central

    Pomés, Anna

    2010-01-01

    The 3-dimensional structure of an allergen defines the accessible parts on the surface of the molecule or epitopes that interact with antibodies. Mapping the antigenic determinants for IgE antibody binding has been pursued through strategies based on the use of overlapping synthetic peptides, recombinant allergenic fragments or unfolded allergens. These approaches led to the identification of mostly linear epitopes and are useful for food allergens that undergo digestion or food processing. For inhaled allergens, conformational epitopes appear to be the primary targets of IgE responses. Knowledge of the molecular structure of allergens alone and in complex with antibodies that interfere with IgE antibody binding is important to understand the immune recognition of B cell-antigenic determinants on allergens and the design of recombinant allergens for immunotherapy. Starting with the molecular cloning and expression of allergens, and with the advent of X-ray crystallography and nuclear magnetic resonance techniques, we have been able to visualize conformational epitopes on allergens. PMID:19940500

  19. Rapid tumor regression in an Asian lung cancer patient following personalized neo-epitope peptide vaccination

    PubMed Central

    Li, Fenge; Chen, Caixia; Ju, Tao; Gao, Junqin; Yan, Jun; Wang, Peng; Xu, Qiang; Hwu, Patrick; Du, Xueming; Lizée, Gregory

    2016-01-01

    ABSTRACT Personalized immunotherapy targeting tumor-specific mutations represents a highly promising approach to cancer treatment. Here, we describe an Asian lung squamous cell carcinoma patient demonstrating frank disease progression following chemotherapy and EGFR inhibitor treatment. Based on tumor mutational profiling and HLA typing, a saline-based multi-epitope peptide vaccine was designed and administered along with topical imiquimod as an adjuvant. Weekly neo-epitope peptide vaccination was followed by a rapid and dramatic regression of multiple lung tumor nodules, while a much larger liver metastasis remained refractory to treatment. Peripheral blood immune monitoring showed that specific cytotoxic T lymphocytes (CTLs) were induced primarily against peptide targets encompassing the widely shared EGFR L858R mutation, particularly one restricted to HLA-A*3101. Immunological targeting of this driver mutation may be of particular benefit to Asian lung cancer patients due to its relatively high prevalence within this patient population. PMID:28123873

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

    PubMed Central

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

    2016-01-01

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

  1. B cell epitope spreading: mechanisms and contribution to autoimmune diseases.

    PubMed

    Cornaby, Caleb; Gibbons, Lauren; Mayhew, Vera; Sloan, Chad S; Welling, Andrew; Poole, Brian D

    2015-01-01

    While a variety of factors act to trigger or initiate autoimmune diseases, the process of epitope spreading is an important contributor in their development. Epitope spreading is a diversification of the epitopes recognized by the immune system. This process happens to both T and B cells, with this review focusing on B cells. Such spreading can progress among multiple epitopes on a single antigen, or from one antigenic molecule to another. Systemic lupus erythematosus, multiple sclerosis, pemphigus, bullous pemphigoid and other autoimmune diseases, are all influenced by intermolecular and intramolecular B cell epitope spreading. Endocytic processing, antigen presentation, and somatic hypermutation act as molecular mechanisms that assist in driving epitope spreading and broadening the immune response in autoimmune diseases. The purpose of this review is to summarize our current understanding of B cell epitope spreading with regard to autoimmunity, how it contributes during the progression of various autoimmune diseases, and treatment options available.

  2. Efficient epitope mapping by bacteriophage {lambda} surface display

    SciTech Connect

    Kuwabara, I.; Maruyama, H.; Zuberi, R.I.

    1997-01-01

    A bacteriophage {lambda} surface expression system, {lambda}foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase I to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries. 25 refs., 4 figs., 1 tab.

  3. Epitope-specificities of HLA antibodies: the effect of epitope structure on Luminex technique-dependent antibody reactivity.

    PubMed

    Resse, Marianna; Paolillo, Rossella; Minucci, Biagio Pellegrino; Cavalca, Francesco; Casamassimi, Amelia; Napoli, Claudio

    2015-04-01

    The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.

  4. Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope.

    PubMed

    Azoitei, Mihai L; Ban, Yih-En Andrew; Julien, Jean-Philippe; Bryson, Steve; Schroeter, Alexandria; Kalyuzhniy, Oleksandr; Porter, Justin R; Adachi, Yumiko; Baker, David; Pai, Emil F; Schief, William R

    2012-01-06

    Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.

  5. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope

    PubMed Central

    1994-01-01

    Mutations that abrogate recognition of a viral epitope by class I- restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with

  6. Broad epitope coverage of a human in vitro antibody library.

    PubMed

    Sivasubramanian, Arvind; Estep, Patricia; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Niles, Rebecca Hurley; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

  7. Vaccines targeting the cancer-testis antigen SSX-2 elicit HLA-A2 epitope-specific cytolytic T cells.

    PubMed

    Smith, Heath A; McNeel, Douglas G

    2011-10-01

    The cancer-testis antigen synovial sarcoma X breakpoint-2 (SSX-2) is a potentially attractive target for tumor immunotherapy based upon its tissue-restricted expression to germline cells and its frequent expression in malignancies. The goal of this study was to evaluate genetic vaccine encoding SSX-2 to prioritize human leukocyte antigen (HLA)-A2-specific epitopes and determine if a DNA vaccine can elicit SSX-2-specific cytotoxic T lymphocytes (CTLs) capable of lysing prostate cancer cells. HLA-A2-restricted epitopes were identified based on their in vitro binding affinity for HLA-A2 and by the ability of a genetic vaccine to elicit peptide-specific CTL in A2/DR1 (HLA-A2.1+/HLA-DR1+/H-2 class I-/class II-knockout) transgenic mice. We found that SSX-2 peptides p41-49 (KASEKIFYV) and p103-111 (RLQGISPKI) had high affinity for HLA-A2 and were immunogenic in vivo; however, peptide p103-111 was immunodominant with robust peptide-specific immune responses elicited in mice vaccinated with a plasmid DNA vaccine encoding SSX-2. Furthermore, p103-111-specific CTLs were able to lyse an HLA-A2+ prostate cancer cell line. The immunodominance of this epitope was found not to be due to a putative HLA-DR1 epitope (p98-112) flanking p103-111. Finally, we demonstrated that SSX-2 epitope-specific CTLs could be detected and cultured from the peripheral blood of HLA-A2+ prostate cancer patients, notably patients with advanced prostate cancer. Overall, we conclude that SSX-2 peptide p103-111 is an immunodominant HLA-A2-restricted epitope, and epitope-specific CD8 T cells can be detected in patients with prostate cancer, suggesting that tolerance to SSX-2 can be circumvented in vivo. Together, these findings suggest that SSX-2 may be a relevant target antigen for prostate cancer vaccine approaches.

  8. Immunization with a peptide containing MHC class I and II epitopes derived from the tumor antigen SIM2 induces an effective CD4 and CD8 T-cell response.

    PubMed

    Kissick, Haydn T; Sanda, Martin G; Dunn, Laura K; Arredouani, Mohamed S

    2014-01-01

    Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2(237-245), was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2(237-245) epitope, and an IL-2 response by CD4 T cells to the SIM2(240-254) epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.

  9. Proof of principle for epitope-focused vaccine design

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  10. Proof of principle for epitope-focused vaccine design

    PubMed Central

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

    2014-01-01

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

  11. Comprehensive mapping infection-enhancing epitopes of dengue pr protein using polyclonal antibody against prM.

    PubMed

    Luo, Yayan; Guo, Xiaolan; Yan, Huijun; Fang, Danyun; Zeng, Gucheng; Zhou, Junmei; Jiang, Lifang

    2015-07-01

    Dengue vaccine development is considered a global public health priority, but the antibody-dependent enhancement (ADE) issues have critically restricted vaccine development. Recent findings have demonstrated that pre-membrane (prM) protein was involved in dengue virus (DENV) infection enhancement. Although the importance of prM antibodies have been well characterized, only a few epitopes in DENV prM protein have ever been identified. In this study, we screened five potential linear epitopes located at positions pr1 (1-16aa), pr3 (13-28aa), pr4 (19-34aa), pr9 (49-64aa), and pr10 (55-70aa) in pr protein using peptide scanning and comprehensive bioinformatics analysis. Then, we found that only pr4 (19-34aa) could elicit high-titer antibodies in Balb/c mice, and this epitope could react with sera from DENV2-infected patients, suggesting that specific antibodies against epitope peptide pr4 were elicited in both DENV-infected mice and human. In addition, our data demonstrated that anti-pr4 sera showed limited neutralizing activity but significant ADE activity toward standard DENV serotypes and imDENV. Hence, it seems responsible to hypothesize that anti-pr4 serum was infection-enhancing antibody and pr4 was infection-enhancing epitope. In conclusion, we characterized a novel infection-enhancing epitope on dengue pr protein, a finding that may provide new insight into the pathogenesis of DENV infection and contribute to dengue vaccine design.

  12. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii.

    PubMed Central

    Saavedra, R; Becerril, M A; Dubeaux, C; Lippens, R; De Vos, M J; Hérion, P; Bollen, A

    1996-01-01

    The ROP2 protein of Toxoplasma gondii possesses immunological and biological properties which have led to its proposal as a vaccine candidate. To identify epitopes recognized by human T cells in the ROP2 antigen, we submitted the sequence of this protein to three reported T-cell epitope prediction algorithms. Three sequences that were predicted by all three methods were selected (sequences 197 to 216, 393 to 410, and 501 to 524), and the corresponding peptides were synthesized. The peptides were first tested in a proliferation assay with a DPw4-restricted, ROP2-specific human T-cell clone, and the peptide corresponding to residues 197 to 216 was shown to stimulate the T-cell clone. The three peptides were further tested in proliferation assays with peripheral blood mononuclear cells from a panel of T. gondii-seropositive and -seronegative individuals. We found that cells from a high proportion of the seropositive donors (64%) recognized at least one of the three peptides. The most frequently recognized ones were peptides 197 to 216 (45%) and 501 to 524 (36%). None of the seronegative donors responded to any peptide. These results show that the ROP2 antigen of T. gondii contains T-cell epitopes recognized by a high percentage of the immune population and further strengthen its potential as a vaccine candidate. PMID:8751939

  13. Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

    PubMed Central

    Upadhya, Rajendra; Kim, Kami; Hogue-Angeletti, Ruth; Weiss, Louis M

    2011-01-01

    Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii. PMID:21352857

  14. Predictions versus high-throughput experiments in T-cell epitope discovery: competition or synergy?

    PubMed

    Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2012-01-01

    Prediction methods as well as experimental methods for T-cell epitope discovery have developed significantly in recent years. High-throughput experimental methods have made it possible to perform full-length protein scans for epitopes restricted to a limited number of MHC alleles. The high costs and limitations regarding the number of proteins and MHC alleles that are feasibly handled by such experimental methods have made in silico prediction models of high interest. MHC binding prediction methods are today of a very high quality and can predict MHC binding peptides with high accuracy. This is possible for a large range of MHC alleles and relevant length of binding peptides. The predictions can easily be performed for complete proteomes of any size. Prediction methods are still, however, dependent on good experimental methods for validation, and should merely be used as a guide for rational epitope discovery. We expect prediction methods as well as experimental validation methods to continue to develop and that we will soon see clinical trials of products whose development has been guided by prediction methods.

  15. Atomic-level mapping of antibody epitopes on a GPCR.

    PubMed

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

  16. Internal epitope tagging informed by relative lack of sequence conservation

    PubMed Central

    Burg, Leonard; Zhang, Karen; Bonawitz, Tristan; Grajevskaja, Viktorija; Bellipanni, Gianfranco; Waring, Richard; Balciunas, Darius

    2016-01-01

    Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function. PMID:27892520

  17. Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18

    PubMed Central

    Zimic, Mirko; Gutiérrez, Andrés Hazaet; Gilman, Robert Hugh; López, César; Quiliano, Miguel; Evangelista, Wilfredo; Gonzales, Armando; García, Héctor Hugo; Sheen, Patricia

    2011-01-01

    Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18. PMID:21738328

  18. Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18.

    PubMed

    Zimic, Mirko; Gutiérrez, Andrés Hazaet; Gilman, Robert Hugh; López, César; Quiliano, Miguel; Evangelista, Wilfredo; Gonzales, Armando; García, Héctor Hugo; Sheen, Patricia

    2011-01-01

    Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18.

  19. Reflections on HLA Epitope-Based Matching for Transplantation

    PubMed Central

    Duquesnoy, Rene J.

    2016-01-01

    HLA antibodies are primary causes of transplant rejection; they recognize epitopes that can be structurally defined by eplets. There are many reviews about HLA epitope-based matching in transplantation. This article describes some personal reflections about epitopes including a historical perspective of HLA typing at the antigen and allele levels, the repertoires of antibody-verified HLA epitopes, the use of HLAMatchmaker in determining the specificities of antibodies tested in different assays, and, finally, possible strategies to control HLA antibody responses. PMID:27965660

  20. Defining the immunogenicity and antigenicity of HLA epitopes is crucial for optimal epitope matching in clinical renal transplantation.

    PubMed

    Kramer, C S M; Roelen, D L; Heidt, S; Claas, F H J

    2017-04-05

    Transplantation of an human leukocyte antigen (HLA) mismatched graft can lead to the development of donor-specific antibodies (DSA), which can result in antibody mediated rejection and graft loss as well as complicate repeat transplantation. These DSA are induced by foreign epitopes present on the mismatched HLA antigens of the donor. However, not all epitopes appear to be equally effective in their ability to induce DSA. Understanding the characteristics of HLA epitopes is crucial for optimal epitope matching in clinical transplantation. In this review, the latest insights on HLA epitopes are described with a special focus on the definition of immunogenicity and antigenicity of HLA epitopes. Furthermore, the use of this knowledge to prevent HLA antibody formation and to select the optimal donor for sensitised transplant candidates will be discussed.

  1. First report on the antibody verification of HLA-ABC epitopes recorded in the website-based HLA Epitope Registry.

    PubMed

    Duquesnoy, R J; Marrari, M; Mulder, A; Sousa, L C D da Mata; da Silva, A S; do Monte, S J H

    2014-06-01

    The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

  2. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  3. Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

    PubMed

    Mishra, Sasmita; Lavelle, Bianca J; Desrosiers, Joe; Ardito, Matt T; Terry, Frances; Martin, William D; De Groot, Anne S; Gregory, Stephen H

    2014-01-01

    Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

  4. Nucleosomal peptide epitopes for nephritis-inducing T helper cells of murine lupus

    PubMed Central

    1996-01-01

    Nucleosome-specific T helper (Th) cells provide major histocompatibility complex class II-restricted, cognate help to nephritogenic antinuclear autoantibody-producing B cells in lupus. However, the lupus Th cells do not respond when components of the nucleosome, such as free DNA or histones, are individually presented by antigen-presenting cells. Thus critical peptide epitopes for the pathogenic Th cells are probably protected during uptake and processing of the native nucleosome particle as a whole. Therefore, herein we tested 145 overlapping peptides spanning all four core histones in the nucleosome. We localized three regions in core histones, one in H2B at amino acid position 10-33 (H2B(10-33)), and two in H4, at position 16- 39 (H4(16-39)) and position 71-94 (H4(71-94)), that contained the peptide epitopes recognized by the pathogenic autoantibody-inducing Th cells of lupus. The peptide autoepitopes also triggered the pathogenic Th cells of (SWR x NZB)F1 lupus mice in vivo to induce the development of severe lupus nephritis. The nucleosomal autoepitopes stimulated the production of Th1-type cytokines, consistent with immunoglobulin IgG2a, IgG2b, and IgG3 being the isotypes of nephritogenic autoantibodies induced in the lupus mice. Interestingly, the Th cell epitopes overlapped with regions in histones that contain B cell epitopes targeted by autoantibodies, as well as the sites where histones contact with DNA in the nucleosome. Identification of the disease-relevant autoepitopes in nucleosomes will help in understanding how the pathogenic Th cells of spontaneous systemic lupus erythematosus emerge, and potentially lead to the development of peptide-based tolerogenic therapy for this major autoimmune disease. PMID:8676066

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

    PubMed

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

    2010-07-19

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

  6. Resistance-associated epitopes of HIV-1C-highly probable candidates for a multi-epitope vaccine.

    PubMed

    Sundaramurthi, Jagadish Chandrabose; Swaminathan, Soumya; Hanna, Luke Elizabeth

    2012-10-01

    Earlier studies have identified a large number of immunogenic epitopes in HIV-1. Efforts are required to prioritize these epitopes in order to identify the best candidates for formulating an effective multi-epitope vaccine for HIV. We modeled 155 known cytotoxic T lymphocyte epitopes of HIV-1 subtype C on the 3D structure of HLA-A*0201, HLA-B*2705, and HLA-B*5101 using MODPROPEP, as these alleles are known to be associated with resistance to HIV/slow progression to AIDS. Thirty-six epitopes were identified to bind to all the three HLA alleles with better binding affinity than the control peptides complexed with each HLA allele but not to any of the HLA alleles reported to be associated with susceptibility to HIV infection/rapid progression to disease. As increase in stability of the epitope-HLA complex results in increased immunogenicity, the short-listed epitopes could be suitable candidates for vaccine development. Twenty of the 36 epitopes were polyfunctional in nature adding to their immunological relevance for vaccine design. Further, 9 of the 20 polyfunctional epitopes were found to bind to all three resistance-associated HLA alleles using an additional method, adding worth to their potential as candidates for a vaccine formulation for HIV-1C.

  7. Influenza, but not HIV-specific CTL epitopes, elicits delayed-type hypersensitivity (DTH) reactions in HIV-infected patients.

    PubMed

    Ruiz-Riol, Marta; Mothe, Beatriz; Gandhi, Rajesh T; Bhardwaj, Nina; Scadden, David T; Sanchez-Merino, Victor; Brander, Christian

    2013-06-01

    The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA-A*02:01-restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed-type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4(+) T-cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope-specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large-scale HIV vaccine trials.

  8. HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

    SciTech Connect

    Tsao, Y.-P.; Lin, J.-Y.; Jan, J.-T.; Leng, C.-H.; Chu, C.-C.; Yang, Y.-C.; Chen, S.-L. . E-mail: showlic@ha.mc.ntu.edu.tw

    2006-05-26

    The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla{sub b}ind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be First identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-{gamma} stimulation of blood CD8{sup +} T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.

  9. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  10. Broad epitope coverage of a human in vitro antibody library

    PubMed Central

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  11. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  12. B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines.

    PubMed

    Blanco, Esther; Cubillos, Carolina; Moreno, Noelia; Bárcena, Juan; de la Torre, Beatriz G; Andreu, David; Sobrino, Francisco

    2013-01-01

    Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD) vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T) or four (B4T) copies of the B-cell epitope from type O FMDV (a widespread circulating serotype) were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFN γ . Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  13. Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: biological validation of the proposed HLA A24 supertype.

    PubMed

    Burrows, Scott R; Elkington, Rebecca A; Miles, John J; Green, Katherine J; Walker, Susan; Haryana, Sofia M; Moss, Denis J; Dunckley, Heather; Burrows, Jacqueline M; Khanna, Rajiv

    2003-08-01

    Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

  14. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    PubMed

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  15. The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing*

    PubMed Central

    Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Henklein, Petra; Sidney, John; Sette, Alessandro; Kloetzel, Peter M.; Mishto, Michele

    2015-01-01

    MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209–217tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201–230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209–217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8+ T cell stimulation in vitro similar to the wtgp100209–217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8+ T cell response also towards N-terminally extended versions of the minimal epitope. PMID:26507656

  16. Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope

    PubMed Central

    1996-01-01

    Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535- 551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Godlewska, Marlena; Czarnocka, Barbara; Gora, Monika

    2012-09-01

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

  19. An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans.

    PubMed Central

    Shirai, M; Okada, H; Nishioka, M; Akatsuka, T; Wychowski, C; Houghten, R; Pendleton, C D; Feinstone, S M; Berzofsky, J A

    1994-01-01

    Several cytotoxic T-lymphocyte (CTL) epitopes have been defined in hepatitis C virus (HCV) proteins. CTL may play an important role in the control of infection by HCV. Here, we identify a highly conserved antigenic site in the HCV core recognized by both murine and human CTL. Spleen cells from mice immunized with a recombinant vaccinia virus expressing the HCV core gene were restimulated in vitro with 11 peptides from the core protein. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 129-144). This conserved epitope was presented by a murine class I major histocompatibility molecule (H-2Dd) to conventional CD4- CD8+ CTL mapped by using transfectants expressing Dd, Ld, or Kd, but was not seen by CTL restricted by H-2b. The murine epitope was mapped to the decapeptide LMGYIPLVGA. The same 16-residue peptide was recognized by CTL from two HCV-seropositive patients but not by CTL from any seronegative donors. CTL from two HLA-A2-positive patients with acute and chronic hepatitides C recognized a 9-residue fragment (DLMGYIPLV) of the peptide presented by HLA-A2 and containing an HLA-A2-binding motif, extending only 1 residue beyond the murine epitope. Therefore, this conserved peptide, seen with murine CTL and human CTL with a very prevalent HLA class I molecule, may be a valuable component of an HCV vaccine against a broad range of HCV isolates. This study demonstrates that the screening for CTL epitopes in mice prior to human study may be useful. PMID:7512163

  20. Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.

    PubMed

    Bashyam, Hema S; Green, Sharone; Rothman, Alan L

    2006-03-01

    Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.

  1. Bypass of carrier-induced epitope-specific suppression using a T-helper epitope.

    PubMed Central

    Sad, S; Rao, K; Arora, R; Talwar, G P; Raghupathy, R

    1992-01-01

    A gonadotropin-releasing hormone (GnRH)-based vaccine is being developed as a method for non-surgical immunotherapy as immunization with this vaccine results in atrophy of the prostate. This vaccine, a conjugate of GnRH and diphtheria toxoid (DT), provides a unique hapten-carrier system for investigating the influence of carrier presensitization on antibody responses to self haptens. In a recent communication we showed that preimmunization with carriers diphtheria toxoid and tetanus toxoid results in a strain-dependent inhibition of anti-GnRH responses in mice and that T cells from carrier-presensitized mice are responsible for anti-haptenic suppression. In the present report we describe a strategy for bypassing DT-induced epitopic suppression using a T-helper epitope from DT. PMID:1383134

  2. Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay

    PubMed Central

    Kang, Mino; Kim, Su Yeon; An, Seong Soo A; Ju, Young Ran

    2013-01-01

    Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid1–42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23–39 and 93–119 in the prion protein were involved in binding to β-amyloid1–40 and 1–42, and monomers of this protein interacted with prion protein residues 93–113 and 123–166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid1–42 at residues 23–40, 104–122 and 159–175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid1–40 and 1–42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease. PMID:23907583

  3. Epitope-vaccine strategy against HIV-1: today and tomorrow.

    PubMed

    Liu, Zuqiang; Xiao, Yi; Chen, Ying-Hua

    2003-01-01

    Vaccines play important roles in preventing infectious diseases caused by different pathogens. However, some pathogens such as HIV-1 challenge current vaccine strategy. Poor immunogenicity and the high mutation rate of HIV-1 make great difficulties in inducing potent immune responses strong enough to prevent infection via vaccination. Epitope-vaccine, which could intensively enhance predefined epitope-specific immune responses, was suggested as a new strategy against HIV-1 and HIV-1 mutation. Epitope-vaccines afford powerful approaches to elicit potent, broad and complete immune protection against not only primary homologous viral isolates but also heterologous viral mutants. Although most studies are still preliminary now, epitope-vaccine as a novel strategy against the AIDS epidemic has great developmental potential. To trigger T-cell-dependent IgG antibody responses and improve affinities of the epitope-specific antibodies, approaches such as recombinant multi-epitope-vaccination and prime-boosting vaccination were suggested. Cellular immune responses, especially CTL responses, could also be elicited and enhanced in addition to humoral immune responses. Developed epitope-vaccines activating both arms of the immune system would benefit prevention and immunotherapy not only against HIV but also other chronic infections.

  4. Building Classifier Ensembles for B-Cell Epitope Prediction

    PubMed Central

    EL-Manzalawy, Yasser; Honavar, Vasant

    2015-01-01

    Identification of B-cell epitopes in target antigens is a critical step in epitope-driven vaccine design, immunodiagnostic tests, and antibody production. B-cell epitopes could be linear, i.e., a contiguous amino acid sequence fragment of an antigen, or conformational, i.e., amino acids that are often not contiguous in the primary sequence but appear in close proximity within the folded 3D antigen structure. Numerous computational methods have been proposed for predicting both types of B-cell epitopes. However, the development of tools for reliably predicting B-cell epitopes remains a major challenge in immunoinformatics. Classifier ensembles a promising approach for combining a set of classifiers such that the overall performance of the resulting ensemble is better than the predictive performance of the best individual classifier. In this chapter, we show how to build a classifier ensemble for improved prediction of linear B-cell epitopes. The method can be easily adapted to build classifier ensembles for predicting conformational epitopes. PMID:25048130

  5. Dissecting Antibodies with Regards to Linear and Conformational Epitopes

    PubMed Central

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

    2015-01-01

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

  6. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6

    PubMed Central

    Wang, Yanhua; Wang, Guangxiang; Cai, Jian Ping

    2016-01-01

    The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents. PMID:27658594

  7. Infection with Trypanosoma cruzi restricts the repertoire of parasite-specific CD8+ T cells leading to immunodominance.

    PubMed

    Tzelepis, Fanny; de Alencar, Bruna C G; Penido, Marcus L O; Claser, Carla; Machado, Alexandre V; Bruna-Romero, Oscar; Gazzinelli, Ricardo T; Rodrigues, Mauricio M

    2008-02-01

    Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.

  8. Identification of cytotoxic T lymphocyte epitopes in dengue virus serotype 1.

    PubMed

    Duan, Zhiliang; Guo, Jianglong; Huang, Xi; Liu, Huifang; Chen, Xinyu; Jiang, Minghua; Wen, Jinsheng

    2015-07-01

    Dengue virus (DENV) has a serious and growing impact on global health and the exact role of DENV-specific CD8(+) T-cells in DENV infection is still uncertain. In the present study, SYFPEITHI algorithm was used to screen the amino acid sequence of Dengue virus serotype 1 (DENV-1) for potential epitopes, and seven putative HLA-A*1101-restricted and five putative HLA-A*2402-restricted epitopes conserved in hundreds of DENV-1 strains were synthesized. The binding affinity of these epitope candidates to corresponding HLA molecules was evaluated using competitive peptide-binding assay. The immunogenicity and specificity of peptides were further tested in HLA-A*1101 transgenic mice, HLA-A*2402 transgenic mice and peripheral blood mononuclear cells (PBMCs) of patients infected with DENV-1. Percentage inhibition (PI) values calculated in competitive peptide-binding assay showed that six peptides (E39-47 PTLDIELLK, NS5(505-513) GVEGEGLHK, NS2b(15-23) SILLSSLLK, NS5(561-569) ALLATSIFK, NS3(99-107) AVEPGKNPK, and NS4b(159-167) VVYDAKFEK) could bind to HLA-A*1101 molecule with high affinity and five peptides (NS3472-480 QYIYMGQPL, NS4a40-48 AYRHAMEEL, NS5(880-888) DYMTSMKRF, NS3(548-556) SYKVASEGF, and NS3(22-30) IYRILQRGL) have a high affinity for HLA-A*2402 molecule. Enzyme-linked immunospot (ELISPOT) results indicated that these high-affinity peptides were recognized by splenocytes of DENV-1-infected transgenic mice and high-affinity peptide-immunized transgenic mice displayed high levels of peptide-specific IFN-γ-secreting cells. In addition, both peptide-pulsed splenocytes and DENV-1-infected splenic monocytes were efficiently killed by these peptide-specific cytotoxic T lymphocytes. Finally, except NS2b(15-23), 10 high-affinity peptides were recognized by PBMCs of patients infected with DENV-1. These identified epitopes would contribute to the understanding of the function of DENV-specific CD8(+) T-cells.

  9. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  10. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

  11. Identification of CD8+ cytotoxic T lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in BALB/c mice

    PubMed Central

    2011-01-01

    Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV. PMID:21619712

  12. Identification of cytotoxic T lymphocyte epitopes on swine viruses: multi-epitope design for universal T cell vaccine.

    PubMed

    Liao, Yu-Chieh; Lin, Hsin-Hung; Lin, Chieh-Hua; Chung, Wen-Bin

    2013-01-01

    Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES.

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

    SciTech Connect

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

    2012-06-28

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

  14. Celiac disease T-cell epitopes from gamma-gliadins: immunoreactivity depends on the genome of origin, transcript frequency, and flanking protein variation

    PubMed Central

    2012-01-01

    Background Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. Results A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. Conclusions Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat

  15. Epitope discovery and their use in peptide based vaccines.

    PubMed

    Dudek, Nadine L; Perlmutter, Patrick; Aguilar, Marie-Isabel; Croft, Nathan P; Purcell, Anthony W

    2010-01-01

    With recent advances in the design and delivery of peptide-based therapeutics there has been a growing interest on the use of peptides in vaccine design. Moreover, functional dissection and proteomic analysis of the immunogenic epitopes of proteins from pathogenic micro-organisms, cancers and self-tissues targeted by autoimmune responses, have broadened the range of target epitopes and given clues to enhancing peptide immunogenicity. Consistent with these observations; peptides can be synthesised with defined chemical modifications to mimic natural epitopes and/or deliberately introduce protease resistant peptide bonds to regulate their processing independent of tissue specific proteolysis and to stabilize these compounds in vivo. We discuss the potential of peptide-based vaccines for the treatment of chronic viral diseases and cancer and review recent developments in the field of epitope discovery and peptide-based vaccines.

  16. Structure of allergens and structure based epitope predictions☆

    PubMed Central

    Dall’Antonia, Fabio; Pavkov-Keller, Tea; Zangger, Klaus; Keller, Walter

    2014-01-01

    The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction. PMID:23891546

  17. Immunogenicity of a multi-epitope DNA vaccine against hantavirus.

    PubMed

    Zhao, Chen; Sun, Ying; Zhao, Yujie; Wang, Si; Yu, Tongtong; Du, Feng; Yang, X Frank; Luo, Enjie

    2012-02-01

    Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.

  18. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  19. Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.

    PubMed

    Tye-Din, Jason A; Stewart, Jessica A; Dromey, James A; Beissbarth, Tim; van Heel, David A; Tatham, Arthur; Henderson, Kate; Mannering, Stuart I; Gianfrani, Carmen; Jewell, Derek P; Hill, Adrian V S; McCluskey, James; Rossjohn, Jamie; Anderson, Robert P

    2010-07-21

    Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

  20. Biology of DNA restriction.

    PubMed Central

    Bickle, T A; Krüger, D H

    1993-01-01

    Our understanding of the evolution of DNA restriction and modification systems, the control of the expression of the structural genes for the enzymes, and the importance of DNA restriction in the cellular economy has advanced by leaps and bounds in recent years. This review documents these advances for the three major classes of classical restriction and modification systems, describes the discovery of a new class of restriction systems that specifically cut DNA carrying the modification signature of foreign cells, and deals with the mechanisms developed by phages to avoid the restriction systems of their hosts. PMID:8336674

  1. Genetics of restrictive cardiomyopathy.

    PubMed

    Sen-Chowdhry, Srijita; Syrris, Petros; McKenna, William J

    2010-04-01

    Restrictive physiology, a severe form of diastolic dysfunction, is characteristically observed in the setting of constrictive pericarditis and myocardial restriction. The latter is commonly due to systemic diseases, some of which are inherited as mendelian traits (eg, hereditary amyloidosis), while others are multifactorial (eg, sarcoidosis). When restrictive physiology occurs as an early and dominant feature of a primary myocardial disorder, it may be termed restrictive cardiomyopathy. In the past decade, clinical and genetic studies have demonstrated that restrictive cardiomyopathy as such is part of the spectrum of sarcomeric disease and frequently coexists with hypertrophic cardiomyopathy in affected families.

  2. Mimotope vaccines: epitope mimics induce anti-cancer antibodies.

    PubMed

    Riemer, Angelika B; Jensen-Jarolim, Erika

    2007-10-31

    Mimotopes are epitope-mimicking structures. When applied for immunizations they induce desired antibody specificities exclusively based on the principle of molecular mimicry. This is important as antibodies directed against tumor-associated antigens may harbor diverse biological effects depending on their epitope specificity. Thus they may inhibit or promote tumor growth. This review gives an update on different vaccination strategies based on the mimotope concept.

  3. Bioinformatic prediction of the epitopes of Echinococcus granulosus antigen 5

    PubMed Central

    Pan, Wei; Chen, De-Sheng; Lu, Yun-Juan; Sun, Fen-Fen; Xu, Hui-Wen; Zhang, Ya-Wen; Yan, Chao; Fu, Lin-Lin; Zheng, Kui-Yang; Tang, Ren-Xian

    2017-01-01

    The aim of the present study was to predict and analyze the secondary structure, and B and T cell epitopes of Echinococcus granulosus antigen 5 (Ag5) using online software in order to investigate its immunogenicity and preliminarily evaluate its potential as an effective antigen peptide vaccine for cystic echinococcosis. The PortParam program was used to analyze molecular weight, the theoretical isoelectric point, instability index and other physicochemical properties. The secondary structure of the Ag5 protein was predicted using Self-Optimized Prediction method With Alignment and the tertiary structure of the Ag5 protein was predicted using 3DLigandSite together with Center for Biological Sequence Analysis Prediction Servers. Furthermore, the Immune Epitope Database software was used to predict B cell epitopes, and T cell epitopes were predicted with the BioInformatics and Molecular Analysis Section and SYFPEITHI programs. The results demonstrated that α-helixes, β-turns, random coils and extended strands account for 23.35, 10.95, 41.32, and 24.38% of the secondary structure of the Ag5 protein, respectively. Ten potential B cell epitopes of Ag5 were identified as the amino acids sequences 27–39, 70–80, 117–130, 146–168, 250–262, 284–293, 339–349, 359–371, 403–412 and 454–462, and seven potential T cell epitopes were identified as the amino acid sequences 52–60, 57–65, 182–190, 231–239, 273–281, 318–326 and 467–475. Thus, ten B cell epitopes and seven T cell epitopes were identified on Ag5, suggesting the strong immunogenicity of this protein, which could be applied to design antigen peptide vaccines for echinococcosis.

  4. Removing T-cell epitopes with computational protein design

    PubMed Central

    King, Chris; Garza, Esteban N.; Mazor, Ronit; Linehan, Jonathan L.; Pastan, Ira; Pepper, Marion; Baker, David

    2014-01-01

    Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function. PMID:24843166

  5. Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes

    PubMed Central

    2010-01-01

    Background Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC). Methods Dendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2. Results DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells. Conclusions These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3522-530 FLAELAYDL, which can be used to monitor HLA-A2

  6. Revealing the anti-HRP epitope in Drosophila and Caenorhabditis.

    PubMed

    Paschinger, Katharina; Rendić, Dubravko; Wilson, Iain B H

    2009-04-01

    Antibodies are very often used as specific cell and/or tissue markers. An example of this is anti-horseradish peroxidase (HRP), an antibody raised against a plant glycoprotein, which was shown some twenty-five years ago to specifically stain neural tissue in an animal, Drosophila melanogaster. This peculiar finding was later expanded to other invertebrate species including Caenorhabditis elegans, which were also shown to bear anti-HRP epitopes. Initial experiments indicated that the epitopes recognised by anti-HRP in invertebrates are of carbohydrate nature. Indeed, more recent experiments have characterised relevant core alpha1-3-fucosylated N-glycan structures that act as epitopes in various model and parasitic organisms. Moreover, a number of enzymes required for the synthesis of such structures have been identified. Over the years, medically-relevant roles of these structures have become apparent as regards allergenicity and immunoregulation. Although major advances have been made in understanding of the underlying mechanisms and structures related to the anti-HRP epitope, the in vivo role of the relevant epitopes in neural and other tissues is yet to be resolved. Current understanding of the anti-HRP epitopes synthesis and their relevance is discussed and elaborated.

  7. T-Cell Epitope Discovery for Therapeutic Cancer Vaccines.

    PubMed

    Krishna, Sri; Anderson, Karen S

    2016-01-01

    The success of recent immune checkpoint blockade trials in solid tumors has demonstrated the tremendous potential of immune-mediated treatment strategies for cancer therapy. These immune therapies activate preexisting cytotoxic CD8(+) T cells (CTL) to selectively target and eradicate malignant cells. In vitro models suggest that these therapies may be more effective in combination with priming of CTL using cancer vaccines. CTL-mediated tumor targeting is achieved by its recognition of tumor antigenic epitopes presented on human leukocyte antigen (HLA) class I molecules by tumor cells. Discovering CTL-antigenic epitopes is therefore central to the design of therapeutic T-cell vaccines and immune monitoring of these complex immunotherapies. However, selecting and monitoring T-cell epitopes remains difficult due to the extensive polymorphism of HLA alleles and the presence of confounding non-immunogenic self-peptides. To overcome these challenges, this chapter presents methodologies for the design of CTL-targeted vaccines using selection of target HLA alleles, novel integrated computational strategies to predict HLA-class I CTL epitopes, and epitope validation methods using short-term ex vivo T-cell stimulation. This strategy results in the improved efficiency for selecting antigenic epitopes for CTL-mediated vaccines and for immune monitoring of tumor antigens.

  8. Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

    PubMed Central

    Croft, Nathan P.; Smith, Stewart A.; Wong, Yik Chun; Tan, Chor Teck; Dudek, Nadine L.; Flesch, Inge E. A.; Lin, Leon C. W.; Tscharke, David C.; Purcell, Anthony W.

    2013-01-01

    Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity. PMID:23382674

  9. Characterization of Conformation-dependent Prion Protein Epitopes*

    PubMed Central

    Kang, Hae-Eun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason; Hunter, Nora; Telling, Glenn C.

    2012-01-01

    Whereas prion replication involves structural rearrangement of cellular prion protein (PrPC), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrPSc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain. PMID:22948149

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

    PubMed Central

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

    2016-01-01

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

  11. Ex vivo detection of adenovirus specific CD4{sup +} T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T{sub HELPER} cells following stem cell transplantation

    SciTech Connect

    Serangeli, Celine; Bicanic, Oliver; Scheible, Michael H.; Lang, Peter; Handgretinger, Rupert

    2010-02-20

    Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4{sup +} T-cell responses against the Hexon-protein, but the frequency of specific T{sub HELPER} cells is extremely low or not detectable ex vivo and preference for different CD4{sup +} T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4{sup +}-responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4{sup +}-proliferation in >50% of individuals, confirmed by intracellular IFN-gamma detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4{sup +} T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4{sup +} T cells for adoptive T-cell transfer against HAdV-infection post SCT.

  12. Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus.

    PubMed

    Johnstone, Carolina; de León, Patricia; Medina, Francisco; Melero, José A; García-Barreno, Blanca; Del Val, Margarita

    2004-11-01

    Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2K(d)-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249-258, which is presented by K(d) as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous K(d) ligand. The CD8(+) T-lymphocyte responses to epitopes F85-93 and F249-258 present in the F protein of RSV Long were found to be strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8(+) T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response.

  13. 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

  14. Type II collagen antibody response is enriched in the synovial fluid of rheumatoid joints and directed to the same major epitopes as in collagen induced arthritis in primates and mice

    PubMed Central

    2014-01-01

    Introduction Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA. Methods Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice. Results Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group. Conclusion CII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation. PMID:25005029

  15. MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes, and is influenced by melanosomes1

    PubMed Central

    Robila, Valentina; Ostankovitch, Marina; Altrich-VanLith, Michelle L.; Theos, Alexander C.; Drover, Sheila; Marks, Michael S.; Restifo, Nicholas; Engelhard, Victor H.

    2009-01-01

    Many human solid tumors express MHC II molecules, and proteins normally localized to melanosomes give rise to MHC II restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHC II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC II molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974

  16. Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

    PubMed Central

    Van Epps, Heather L.; Schmaljohn, Connie S.; Ennis, Francis A.

    1999-01-01

    Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4+ and CD8+ N-specific cell lines from two donors and CD4+ G1-specific cell lines from a third donor. All CD8+ cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus. PMID:10364276

  17. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.

  18. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

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

    2016-01-01

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

  19. Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens.

    PubMed

    Wang, Jian; Lin, Yahui; Cai, Pengfei; Wang, Heng

    2011-01-01

    The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines.

  20. Bcipep: A database of B-cell epitopes

    PubMed Central

    Saha, Sudipto; Bhasin, Manoj; Raghava, Gajendra PS

    2005-01-01

    Background Bcipep is a database of experimentally determined linear B-cell epitopes of varying immunogenicity collected from literature and other publicly available databases. Results The current version of Bcipep database contains 3031 entries that include 763 immunodominant, 1797 immunogenic and 471 null-immunogenic epitopes. It covers a wide range of pathogenic organisms like viruses, bacteria, protozoa, and fungi. The database provides a set of tools for the analysis and extraction of data that includes keyword search, peptide mapping and BLAST search. It also provides hyperlinks to various databases such as GenBank, PDB, SWISS-PROT and MHCBN. Conclusion A comprehensive database of B-cell epitopes called Bcipep has been developed that covers information on epitopes from a wide range of pathogens. The Bcipep will be source of information for investigators involved in peptide-based vaccine design, disease diagnosis and research in allergy. It should also be a promising data source for the development and evaluation of methods for prediction of B-cell epitopes. The database is available at . PMID:15921533

  1. Recombinant expression and epitope mapping of grass pollen allergens.

    PubMed

    Suphioglu, C; Smith, P M; Ong, E K; Knox, R B; Singh, M B

    1996-01-01

    We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

  2. Chronic lymphocytic choriomeningitis virus infection actively down-regulates CD4+ T cell responses directed against a broad range of epitopes.

    PubMed

    Mothé, Bianca R; Stewart, Barbara S; Oseroff, Carla; Bui, Huynh-Hoa; Stogiera, Stephanie; Garcia, Zacarias; Dow, Courtney; Rodriguez-Carreno, Maria Pilar; Kotturi, Maya; Pasquetto, Valerie; Botten, Jason; Crotty, Shane; Janssen, Edith; Buchmeier, Michael J; Sette, Alessandro

    2007-07-15

    Activation of CD4(+) T cells helps establish and sustain CD8(+) T cell responses and is required for the effective clearance of acute infection. CD4-deficient mice are unable to control persistent infection and CD4(+) T cells are usually defective in chronic and persistent infections. We investigated the question of how persistent infection impacted pre-existing lymphocytic choriomeningitis virus (LCMV)-specific CD4(+) T cell responses. We identified class II-restricted epitopes from the entire set of open reading frames from LCMV Armstrong in BALB/c mice (H-2(d)) acutely infected with LCMV Armstrong. Of nine epitopes identified, six were restricted by I-A(d), one by I-E(d) and two were dually restricted by both I-A(d) and I-E(d) molecules. Additional experiments revealed that CD4(+) T cell responses specific for these epitopes were not generated following infection with the immunosuppressive clone 13 strain of LCMV. Most importantly, in peptide-immunized mice, established CD4(+) T cell responses to these LCMV CD4 epitopes as well as nonviral, OVA-specific responses were actively suppressed following infection with LCMV clone 13 and were undetectable within 12 days after infection, suggesting an active inhibition of established helper responses. To address this dysfunction, we performed transfer experiments using both the Smarta and OT-II systems. OT-II cells were not detected after clone 13 infection, indicating physical deletion, while Smarta cells proliferated but were unable to produce IFN-gamma, suggesting impairment of the production of this cytokine. Thus, multiple mechanisms may be involved in the impairment of helper responses in the setting of early persistent infection.

  3. In silico identification of common epitopes from pathogenic mycobacteria.

    PubMed

    de la Caridad Addine Ramírez, Bárbara; Marrón, Reynel; Calero, Rommel; Mirabal, Mayelin; Ramírez, Juan Carlos; Sarmiento, María E; Norazmi, Mohd Nor; Acosta, Armando

    2013-01-01

    An in silico study was carried out to identify antigens for their possible collective use as vaccine candidates against diseases caused by different classes of pathogenic mycobacteria with significant clinical relevance. The genome sequences of the relevant causative agents were used in order to search for orthologous genes among them. Bioinformatics tools permitted us to identify several conserved sequences with 100% identity with no possibility of cross-reactivity to the normal flora and human proteins. Nine different proteins were characterized using the strain H37Rv as reference and taking into account their functional category, their in vivo expression and subcellular location. T and B cell epitopes were identified in the selected sequences. Theoretical prediction of population coverage was calculated for individual epitopes as well as their combinations. Several identical sequences, belonging to six proteins containing T and B cell epitopes which are not present in selected microorganisms of the normal microbial flora or in human proteins were obtained.

  4. Common food allergens and their IgE-binding epitopes.

    PubMed

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  5. In silico identification of common epitopes from pathogenic mycobacteria

    PubMed Central

    2013-01-01

    An in silico study was carried out to identify antigens for their possible collective use as vaccine candidates against diseases caused by different classes of pathogenic mycobacteria with significant clinical relevance. The genome sequences of the relevant causative agents were used in order to search for orthologous genes among them. Bioinformatics tools permitted us to identify several conserved sequences with 100% identity with no possibility of cross-reactivity to the normal flora and human proteins. Nine different proteins were characterized using the strain H37Rv as reference and taking into account their functional category, their in vivo expression and subcellular location. T and B cell epitopes were identified in the selected sequences. Theoretical prediction of population coverage was calculated for individual epitopes as well as their combinations. Several identical sequences, belonging to six proteins containing T and B cell epitopes which are not present in selected microorganisms of the normal microbial flora or in human proteins were obtained. PMID:23458668

  6. Antibody specific epitope prediction-emergence of a new paradigm.

    PubMed

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data.

  7. Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof

    DOEpatents

    Stupp, Samuel I.; Guler, Mustafa O.

    2008-11-18

    Branched peptide amphiphilic compounds incorporating one or residues providing a pendant amino group for coupling one or more epitope sequences thereto, such compounds and related compositions for enhanced epitope presentation.

  8. Major histocompatibility complex conformational epitopes are peptide specific

    PubMed Central

    1992-01-01

    Serologically distinct forms of H-2Kb are stabilized by loading cells expressing "empty" class I major histocompatibility complex (MHC) molecules with different H-2Kb binding peptides. The H-2Kb epitope recognized by monoclonal antibody (mAb) 28.8.6 was stabilized by ovalbumin (OVA) (257-264) and murine cytomegalovirus (MCMV) pp89 (168- 176) peptides, but not by vesicular stomatic virus nucleoprotein (VSV NP) (52-59) and influenza NP (Y345-360) peptides. The H-2Kb epitope recognized by mAb 34.4.20 was stabilized by VSV NP (52-59) peptide but not by OVA (257-264), MCMV pp89 (168-176), or influenza NP (Y345-360) peptides. Immunoprecipitation of H-2Kb molecules from normal cells showed that 28.8.6 and 34.4.20 epitopes were only present on a subset of all conformationally reactive H-2Kb molecules. Using alanine- substituted derivatives of the VSV peptide, the 28.8.6 epitope was completely stabilized by substitution of the first residue and partially stabilized by substitution of the third or the fifth residues in the peptides. These results indicate that distinct conformational MHC epitopes are dependent on the specific peptide that occupies the antigenic peptide binding groove on individual MHC molecules. The changes in MHC epitopes observed may also be important in understanding the diversity of T cell receptors used in an immune response and the influence of peptides on development of the T cell repertoire. PMID:1281212

  9. Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Cappelletti, Francesca; Clementi, Massimo; Burioni, Roberto

    2014-01-01

    The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results.

  10. The value of HIV protective epitope research for informed vaccine design against diverse viral pathogens.

    PubMed

    Kramer, Victor G; Byrareddy, Siddappa N

    2014-08-01

    The success of vaccine regimens against viral pathogens hinges on the elicitation of protective responses. Hypervariable pathogens such as HIV avoid neutralization by masking protective epitopes with more immunogenic decoys. The identification of protective, conserved epitopes is crucial for future vaccine candidate design. The strategies employed for identification of HIV protective epitopes will also aid towards rational vaccine design for other viral pathogens.

  11. Immunogenicity and specificity of the candidate multi-epitope-vaccines against HIV-1.

    PubMed

    Lu, Y; Ding, J; Chen, Y H

    2001-11-01

    The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)2-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.

  12. Enhancement of the antibody response to flavivirus B-cell epitopes by using homologous or heterologous T-cell epitopes.

    PubMed Central

    Roehrig, J T; Johnson, A J; Hunt, A R; Beaty, B J; Mathews, J H

    1992-01-01

    We have been investigating the T-helper (Th)-cell response to the flavivirus envelope (E) glycoprotein. In our studies with Murray Valley encephalitis (MVE) virus, we previously identified synthetic peptides capable of priming Th lymphocytes for an in vitro antivirus proliferative response (J. H. Mathews, J. E. Allan, J. T. Roehrig, J. R. Brubaker, and A. R. Hunt, J. Virol. 65:5141-5148, 1991). We have now characterized in vivo Th-cell priming activity of one of these peptides (MVE 17, amino acids 356 to 376) and an analogous peptide derived from the E-glycoprotein sequence of the dengue (DEN) 2, Jamaica strain (DEN 17, amino acids 352 to 368). This DEN peptide also primed the Th-cell compartment in BALB/c mice, as measured by in vitro proliferation and interleukin production. The failure of some MVE and DEN virus synthetic peptides to elicit an antibody response in BALB/c mice could be overcome if a Th-cell epitope-containing peptide was included in the immunization mixture. A more detailed analysis of the structural interactions between Th-cell and B-cell epitope donor peptides revealed that the peptides must be linked to observe the enhanced antibody response. Blockage or deletion of the free cysteine residue on either peptide abrogated the antibody response. The most efficient T-B-cell epitope interaction occurred when the peptides were colinearly synthesized. These Th-cell-stimulating peptides were also functional with the heterologous B-cell epitope-containing peptides. The Th-cell epitope on DEN 17 was more potent than the Th-cell epitope on MVE 17. PMID:1374807

  13. Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

    PubMed Central

    Mishto, Michele; Bellavista, Elena; Ligorio, Claudia; Textoris-Taube, Kathrin; Santoro, Aurelia; Giordano, Mara; D'Alfonso, Sandra; Listì, Florinda; Nacmias, Benedetta; Cellini, Elena; Leone, Maurizio; Grimaldi, Luigi M.E.; Fenoglio, Chiara; Esposito, Federica; Martinelli-Boneschi, Filippo; Galimberti, Daniela; Scarpini, Elio; Seifert, Ulrike; Amato, Maria Pia; Caruso, Calogero; Foschini, Maria P.; Kloetzel, Peter M.; Franceschi, Claudio

    2010-01-01

    Background Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. Methodology/Principal Findings Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111–119. Conclusion/Significance The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis. PMID:20174631

  14. Energy restriction and potential energy restriction mimetics.

    PubMed

    Nikolai, Sibylle; Pallauf, Kathrin; Huebbe, Patricia; Rimbach, Gerald

    2015-12-01

    Energy restriction (ER; also known as caloric restriction) is the only nutritional intervention that has repeatedly been shown to increase lifespan in model organisms and may delay ageing in humans. In the present review we discuss current scientific literature on ER and its molecular, metabolic and hormonal effects. Moreover, criteria for the classification of substances that might induce positive ER-like changes without having to reduce energy intake are summarised. Additionally, the putative ER mimetics (ERM) 2-deoxy-d-glucose, metformin, rapamycin, resveratrol, spermidine and lipoic acid and their suggested molecular targets are discussed. While there are reports on these ERM candidates that describe lifespan extension in model organisms, data on longevity-inducing effects in higher organisms such as mice remain controversial or are missing. Furthermore, some of these candidates produce detrimental side effects such as immunosuppression or lactic acidosis, or have not been tested for safety in long-term studies. Up to now, there are no known ERM that could be recommended without limitations for use in humans.

  15. Antibodies against HLA-DP recognize broadly expressed epitopes.

    PubMed

    Simmons, Daimon P; Kafetzi, Maria L; Wood, Isabelle; Macaskill, Peter C; Milford, Edgar L; Guleria, Indira

    2016-12-01

    HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies.

  16. Mast Cells Produce a Unique Chondroitin Sulfate Epitope

    PubMed Central

    Farrugia, Brooke L.; Whitelock, John M.; O’Grady, Robert; Caterson, Bruce; Lord, Megan S.

    2015-01-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells. PMID:26586669

  17. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    PubMed

    Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

    2016-02-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.

  18. [TEpredict: software for T-cell epitope prediction].

    PubMed

    Antonets, D V; Maksiutov, A Z

    2010-01-01

    A program named TEpredict was developed for T-cell epitope prediction. Original models for T-cell epitope prediction were constructed by means of Partial Least Squares regression method on the basis of data, extracted from the IEDB (Immune Epitope Database)--the most complete resource of experimental peptide-MHC binding data known to date. TEpredict is also able to predict proteasomal processing of protein antigens, and the ability of produced oligopeptides to bind to TAP (Tansporters Associated with Processing). TEpredict could exclude peptides, shearing local similarity with human proteins, from the set of predicted T-cell epitopes. It is also able to estimate expected population coverage by selected peptides, using known HLA allele genotypic frequencies data. The majority of produced models demonstrated high sensitivity of predictions (0.50-0.80) concurrent with high specificity (0.75-0.99). TEpredict was shown to be highly competitive or even superior in comparison with such programs as ProPred1, SVRMHC, SVMHC and SYFPEITHI. TEpredict demonstrated high quality of predictions and we expect that it could become a useful tool in the development ofpolyepitope vaccines against dangerous human pathogens, including HIV, influenza etc. The program and its source code could be freely downloaded from the project web-site: http://tepredict.sourceforge.net.

  19. In vivo immunogenicity of Tax(11-19) epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine.

    PubMed

    Sagar, Divya; Masih, Shet; Schell, Todd; Jacobson, Steven; Comber, Joseph D; Philip, Ramila; Wigdahl, Brian; Jain, Pooja; Khan, Zafar K

    2014-05-30

    Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund's adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses.

  20. Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy

    PubMed Central

    Hussain, R; Dockrell, H M; Chiang, T J

    1999-01-01

    Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (ρ=0·748; P < 0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n = 26) or tuberculoid form (BT/TT, n = 39) of the disease as well as in healthy households (HC, n = 14) and endemic controls (EC = 19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41–55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded. PMID:10233750

  1. In vivo immunogenicity of Tax 11-19 epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine

    PubMed Central

    Sagar, Divya; Masih, Shet; Schell, Todd; Jacobson, Steven; Comber, Joseph D.; Philip, Ramila; Wigdahl, Brian; Jain, Pooja; Khan, Zafar K.

    2014-01-01

    Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund’s adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8 T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses. PMID:24739247

  2. Candidate multi-epitope vaccines in aluminium adjuvant induce high levels of antibodies with predefined multi-epitope specificity against HIV-1.

    PubMed

    Ding, J; Lu, Y; Chen, Y

    2000-10-01

    Some neutralizing epitopes on HIV-1 envelope proteins were identified to induce antibodies which could effectively inhibit the infection of different strains in vitro. But only very low levels of these antibodies were determined in the HIV-1 infected individuals. To increase the levels of protective antibodies in vivo, we suggested multi-epitope vaccine as a new strategy to induce high level of neutralization antibodies with predefined multi-epitope specificity. A synthesized epitope peptide MP (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD) containing three neutralizing epitopes (GPGRAFY, ELDKWA, RILAVERYLKD) was conjugated to carrier protein KLH, and then used for immunization in mouse together with aluminium adjuvant or Freund's adjuvant (FA). The candidate MP-KLH multi-epitope vaccine in aluminium adjuvant could induce antibody response very strongly to the epitope peptide C-(RILAVERYLKD-G)2 and the immunosuppressive peptide (P1) (LQARILAVERYLKDQQL) (antibody titer: 1:51200), strongly to the epitope peptide C-(ELDKWA-G)4 and the C-domain peptide (P2) (1:12800), and moderately to the epitope peptide C-(GPGRAFY)4 and the V3 loop peptide (1:1600). The immunoblotting analysis demonstrated that the antibodies in sera could recognize P1, P2, V3 loop peptides and rsgp41 (aa 539-684). These results are similar with that in the case of PI-BSA in FA, and suggest that the multi-epitope vaccine in aluminium could induce high levels of antibodies of predefined multi-epitope specificity, which provides experimental evidence for the new strategy to develop an effective neutralizing antibody-based multi-epitope vaccine against HIV-1.

  3. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  4. Alphavirus-Specific Cytotoxic T Lymphocytes Recognize a Cross-Reactive Epitope from the Capsid Protein and Can Eliminate Virus from Persistently Infected Macrophages

    PubMed Central

    Linn, May La; Mateo, L.; Gardner, J.; Suhrbier, A.

    1998-01-01

    Persistent alphavirus infections in synovial and neural tissues are believed to be associated with chronic arthritis and encephalitis, respectively, and represent likely targets for CD8+ αβ cytotoxic T lymphocytes (CTL). Here we show that the capsid protein is a dominant target for alphavirus-specific CTL in BALB/c mice and that capsid-specific CTL from these mice recognize an H-2Kd restricted epitope, QYSGGRFTI. This epitope lies in the highly conserved region of the capsid protein, and QYSGGRFTI-specific CTL were cross reactive across a range of Old World alphaviruses. In vivo the acute primary viraemia of these highly cytopathic viruses was unaffected by QYSGGRFTI-specific CTL. However, in vitro these CTL were able to completely clear virus from macrophages persistently and productively infected with the arthrogenic alphavirus Ross River virus. PMID:9573286

  5. Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus.

    PubMed

    Parvege, Md Masud; Rahman, Monzilur; Nibir, Yead Morshed; Hossain, Mohammad Shahnoor

    2016-04-01

    Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings.

  6. An Unstable Th Epitope of P. falciparum Fosters Central Memory T Cells and Anti-CS Antibody Responses

    PubMed Central

    Parra-López, Carlos A.; Bernal-Estévez, David; Vargas, Luis Eduardo; Pulido-Calixto, Carolina; Salazar, Luz Mary; Calvo-Calle, J. Mauricio; Stern, Lawrence J.

    2014-01-01

    Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRβ1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed. PMID:24983460

  7. An unstable Th epitope of P. falciparum fosters central memory T cells and anti-CS antibody responses.

    PubMed

    Parra-López, Carlos A; Bernal-Estévez, David; Yin, Liusong; Vargas, Luis Eduardo; Pulido-Calixto, Carolina; Salazar, Luz Mary; Calvo-Calle, J Mauricio; Stern, Lawrence J

    2014-01-01

    Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRβ1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed.

  8. MUC-1 Tumor Antigen Agonist Epitopes for Enhancing T-cell Responses to Human Tumors | NCI Technology Transfer Center | TTC

    Cancer.gov

    Scientists at NIH have identified 7 new agonist epitopes of the MUC-1 tumor associated antigen. Compared to their native epitope counterparts, peptides reflecting these agonist epitopes have been shown to enhance the generation of human tumor cells, which in turn have a greater ability to kill human tumor cells endogenously expressing the native MUC-1 epitope.

  9. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    PubMed Central

    Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  10. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    PubMed

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  11. Two human antibodies reacting with different epitopes on integrin beta 3 of platelets and endothelial cells.

    PubMed

    Jallu, V; Diaz-Ricart, M; Ordinas, A; Pico, M; Vezon, G; Nurden, A T

    1994-06-15

    , autoantibody activity to the residual GP IIIa was detected in both cases. Thus, patients (AF) and (EBV) have developed anti-GP-IIIa antibodies with restricted and distinct epitopes but recognizing self antigens.

  12. Perioperative Fluid Restriction

    PubMed Central

    Bleier, Joshua I.S.; Aarons, Cary B.

    2013-01-01

    Perioperative fluid management of the colorectal surgical patient has evolved significantly over the last five decades. Older notions espousing aggressive hydration have been shown to be associated with increased complications. Newer data regarding fluid restriction has shown an association with improved outcomes. Management of perioperative fluid administration can be considered in three primary phases: In the preoperative phase, data suggests that avoidance of preoperative bowel preparation and avoidance of undue preoperative dehydration can improve outcomes. Although the type of intraoperative fluid given does not have a significant effect on outcome, data do suggest that a restrictive fluid regimen results in improved outcomes. Finally, in the postoperative phase of fluid management, a fluid-restrictive regimen, coupled with early enteral feeding also seems to result in improved outcomes. PMID:24436675

  13. Restricting Grammatical Complexity

    ERIC Educational Resources Information Center

    Frank, Robert

    2004-01-01

    Theories of natural language syntax often characterize grammatical knowledge as a form of abstract computation. This paper argues that such a characterization is correct, and that fundamental properties of grammar can and should be understood in terms of restrictions on the complexity of possible grammatical computation, when defined in terms of…

  14. Calorie restriction and stroke

    PubMed Central

    2011-01-01

    Stroke, a major cause of disability and mortality in the elderly, occurs when a cerebral blood vessel is occluded or ruptured, resulting in ischemic damage and death of brain cells. The injury mechanism involves metabolic and oxidative stress, excitotoxicity, apoptosis and inflammatory processes, including activation of glial cells and infiltration of leukocytes. In animal models, dietary energy restriction, by daily calorie reduction (CR) or intermittent fasting (IF), extends lifespan and decreases the development of age-related diseases. Dietary energy restriction may also benefit neurons, as suggested by experimental evidence showing that CR and IF protect neurons against degeneration in animal models. Recent findings by our group and others suggest the possibility that dietary energy restriction may protect against stroke induced brain injury, in part by inducing the expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF); protein chaperones, including heat shock protein 70 (Hsp70) and glucose regulated protein 78 (GRP78); antioxidant enzymes, such as superoxide dismutases (SOD) and heme oxygenase-1 (HO-1), silent information regulator T1 (SIRT1), uncoupling proteins and anti-inflammatory cytokines. This article discusses the protective mechanisms activated by dietary energy restriction in ischemic stroke. PMID:21910904

  15. Identification of novel human aggrecan T cell epitopes in HLA-B27 transgenic mice associated with spondyloarthropathy.

    PubMed

    Kuon, Wolfgang; Kuhne, Maren; Busch, Dirk H; Atagunduz, Pamir; Seipel, Martina; Wu, Peihua; Morawietz, Lars; Fernahl, Gabriele; Appel, Heiner; Weiss, Elisabeth H; Krenn, Veit; Sieper, Joachim

    2004-10-15

    The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.

  16. A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

    PubMed Central

    Yogev, D; Menaker, D; Strutzberg, K; Levisohn, S; Kirchhoff, H; Hinz, K H; Rosengarten, R

    1994-01-01

    We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed. Images PMID:7523302

  17. A novel DNA vaccine containing four mimicry epitopes for gastric cancer.

    PubMed

    Chen, Yu; Wu, Kaichun; Guo, Changcun; Liu, Changjiang; Han, Shuang; Lin, Tao; Ning, Xiaoxuan; Shi, Rui; Shi, Yongquan; Fan, Daiming

    2005-03-01

    Gastric cancer is one of the most common malignant tumors in China. This paper focuses on the development of a DNA vaccine containing four mimotopes of MG7Ag for gastric cancer (multi-epitope vaccine). By inoculating BALB/c mice, the vaccine was characterized and compared with a similar vaccine containing only one mimotope (mono-epitope vaccine) and other controls. Cellular ELISA indicated that serum titer of antibody against MG7Ag was significantly higher in mice immunized with the multi-epitope vaccine than that in the group immunized with the mono-epitope vaccine (0.8627 vs 0.6754, P < 0.05). And ELISPOT assay showed that the number of INF-gamma spots induced by multi-epitope vaccine was significantly larger than that of the group immunized with mono-epitope vaccine(93.3 vs 70.7, P < 0.05). Two weeks after tumor challenge, the weight of tumor in each mouse was evaluated, and the tumor masses formed in the mice immunized with multi-epitope vaccine were markedly smaller than those formed in the mice immunized with mono-epitope vaccine. These studies demonstrated that both humoral and cellular response were induced by the two vaccines and the efficiency of multi-epitope vaccine is stronger than that of the mono-epitope vaccine.

  18. Fine mapping of sequential neutralization epitopes on the subunit protein VP8 of human rotavirus.

    PubMed Central

    Kovacs-Nolan, Jennifer; Yoo, Dongwan; Mine, Yoshinori

    2003-01-01

    The epitopes of the HRV (human rotavirus), especially those involved in virus neutralization, have not been determined in their entirety, and would have significant implications for HRV vaccine development. In the present study, we report on the epitope mapping and identification of sequential neutralization epitopes, on the Wa strain HRV subunit protein VP8, using synthetic overlapping peptides. Polyclonal antibodies against recombinant Wa VP8 were produced previously in chicken, and purified from egg yolk, which showed neutralizing activity against HRV in vitro. Overlapping VP8 peptide fragments were synthesized and probed with the anti-VP8 antibodies, revealing five sequential epitopes on VP8. Further analysis suggested that three of the five epitopes detected, M1-L10, I55-D66 and L223-P234, were involved in virus neutralization, indicating that sequential epitopes may also be important for the HRV neutralization. The interactions of the antibodies with the five epitopes were characterized by an examination of the critical amino acids involved in antibody binding. Epitopes comprised primarily of hydrophobic amino acid residues, followed by polar and charged residues. The more critical amino acids appeared to be located near the centre of the epitopes, with proline, isoleucine, serine, glutamine and arginine playing an important role in the binding of antibody to the VP8 epitopes. PMID:12901721

  19. Bioinformatic prediction of the antigenic epitopes of recombinant ferritin of Echinococcus granulosus.

    PubMed

    Liu, Xuelei; Zhao, Hui; Cao, Wenyan; Liu, Yumei; Zhang, Chuntao; Lan, Xi; Peng, Shanshan; Wen, Hao; Ding, Jianbing; Ma, Xiumin

    2016-01-01

    Echinococcosis is a zoonotic parasitic disease affecting humans and other mammals, which is mainly caused Echinococcus at larval stages. It is predominantly endemic in Chinese pasture regions, including Xinjiang, Qinghai, Gansu and Ningxia. The aim of the present study was to predict the T‑ and B‑combined epitopes of Echinococcus granulosus (Eg). ferritin, and to analyze its secondary structure using online software. Prediction of the T‑ and B‑combined epitopes of Eg. ferritin was performed using IEDB, SYFPEITHI and LEPS software, which are used to identify common areas of T‑ and B‑cells. The results of the present study identified several potential antigenic epitopes of Eg. ferritin, including seven B‑cell antigen epitope amino acid sequences with high values: 8‑16, 54‑61, 70‑75, 80‑90, 103‑109, 117‑124 and 167‑173; and four T‑cell antigen epitope amino acid sequences with high values: 85‑93, 105‑113, 133‑141 and 157‑165. Furthermore, a combined epitope region comprising an 105‑109 amino acid sequence was identified. In conclusion, using bioinformatic methods, the present study confirmed the existence of Eg. ferritin on four T‑cell antigen epitopes, seven B‑cell antigen epitopes, and one T‑ and B‑combined epitope region. These findings provide significant information for further investigation of the antigenicity of Eg. ferritin and the development of highly efficient epitope vaccines.

  20. Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library.

    PubMed

    Richter, W; Northemann, W; Müller, M; Böhm, B O

    1996-04-01

    Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2. Using an epitope cDNA library we mapped the MICA 2 epitope to a contiguous stretch of 26 amino acids (506-531) in the C-terminus of GAD65. Neither blocking experiments with synthetic peptides nor analysis of overlapping decapeptides expressed as fusion proteins allowed us to further narrow down the epitope to the typical size of linear epitopes of 6-8 amino acids. We suggest that a miniconformational epitope provided by amino acids 506-531 is recognized by MICA 2, which withstands SDS gel electrophoresis without destruction or partially refolds during the Western blot procedure. A sequence homology with human heat shock protein 60 (HSP60) maps to this region of GAD65 but no cross-reactivity of MICA 2 with HSP60 occurred. Our data demonstrate that reactivity of an antibody in Western blots does not necessarily define a classic linear epitope of 6-8 amino acids and describe a new autoreactive epitope in GAD65 different from those reported for sera from patients with SMS.

  1. Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

    PubMed Central

    Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O.; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen

    2013-01-01

    Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR. PMID:24244326

  2. Epitope predictions indicate the presence of two distinct types of epitope-antibody-reactivities determined by epitope profiling of intravenous immunoglobulins.

    PubMed

    Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen

    2013-01-01

    Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.

  3. [Common epitopes of protein antigens in Neisseria and Moraxella].

    PubMed

    Goncharenko, A V; Filatova, T N; Padiukov, L N; Baturo, A P

    2000-01-01

    Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.

  4. Identification of a major continuous epitope of human alpha crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

  5. A General Method to Discover Epitopes from Sera

    PubMed Central

    Whittemore, Kurt; Johnston, Stephen Albert; Sykes, Kathryn; Shen, Luhui

    2016-01-01

    Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen

  6. Differential expression of proteoglycan epitopes by ovine intervertebral disc cells

    PubMed Central

    MELROSE, JAMES; SMITH, SUSAN; GHOSH, PETER

    2000-01-01

    The alginate bead culture system has been utilised by several groups to examine the in vitro proteoglycan (PG) metabolism of chondrocytes and intervertebral disc cells, but the nature of the PGs produced has not been examined in detail. This is largely due to the difficulty of separating the anionically charged sodium alginate support matrix from PGs which are similarly charged. In the present study ovine annulus fibrosus, transitional zone and nucleus pulposus cells were dissociated enzymatically from their respective matrices by sequential digestion with pronase/clostridial collagenase and DNAase and then cultured in alginate beads for 10 d. The beads were solubilised and subjected to DEAE Sepharose CL6B anion exchange chromatography to separate the sodium alginate bead support matrix material quantitatively from the disc cell PGs. The alginate free bead PGs were then subjected to composite agarose polyacrylamide gel electrophoresis to resolve PG populations and the PGs were transferred to nitrocellulose membranes by semidry electroblotting. The PGs were identified by probing the blots with a panel of antibodies to defined PG core protein and glycosaminoglycan side chain epitopes. Alginate beads of disc cells were also embedded in paraffin wax and 4μm sections cut to immunolocalise decorin, biglycan, versican, and the 7-D-4 PG epitope within the beads. Decorin and biglycan had similar distributions in the beads, being localised on the cell surface whereas versican and the 7-D-4 PG epitope were immunolocalised interterritoriarly. This study is the first to demonstrate that ovine disc cells synthesise versican in alginate bead culture. Furthermore the immunoblotting studies also showed that a proportion of the 7-D-4 PG epitope was colocalised with versican. PMID:11005711

  7. Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

    PubMed Central

    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. PMID:25343355

  8. HLA-A2-Restricted Protection against Lethal Lymphocytic Choriomeningitis▿ †

    PubMed Central

    Botten, Jason; Whitton, J. Lindsay; Barrowman, Polly; Sidney, John; Whitmire, Jason K.; Alexander, Jeff; Ting, Joey P. C.; Bui, Huynh-Hoa; Sette, Alessandro; Buchmeier, Michael J.

    2007-01-01

    The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8+ T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides—nucleoprotein NP69-77, glycoprotein precursor GPC10-18, GPC447-455, and zinc-binding protein Z49-58—that displayed high-affinity binding (≤275 nM) to HLA-A*0201, induced CD8+ T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC447-455), after peptide immunization of HLA-A*0201 mice, induced CD8+ T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV. PMID:17166907

  9. Stochastic humoral expression of human growth hormone epitopes.

    PubMed Central

    Etcheverrigaray, M; Paladini, A C; Retegui, L A

    1988-01-01

    Competition experiments between insolubilized monoclonal antibodies (mAb) and polyclonal antisera has led to the description of the humoral expression of human growth hormone (hGH) epitopes. This study was carried out with sera from mice and hamsters submitted to different immunization schedules: chronic administration of the antigen, secondary response and conventional hyperimmunization. The results indicated the absence of a unique immunodominant epitope in hGH; a significant individual variation of antibody (Ab) population titres with time; changes with time in the relative proportion of one Ab population with respect to the others; and the occurrence of Ab enhancing the 125I-hGH binding to five mAb depending upon the individuals and the time of immunization. Heterocliticity towards non-human GH was also detected. Although most of the animals showed cross-reacting Ab, two out of 12 mice, chronically injected, developed heteroclitic Ab. The data suggest that the humoral response to different epitopes of a protein antigen during the maturation of the immune response is a stochastic process leading to transient humoral immunodominance, enhancing Ab populations and heterocliticity, depending upon individual characteristics, either in outbred or inbred populations. PMID:2452789

  10. Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages.

    PubMed

    Stucki, David; Brites, Daniela; Jeljeli, Leïla; Coscolla, Mireia; Liu, Qingyun; Trauner, Andrej; Fenner, Lukas; Rutaihwa, Liliana; Borrell, Sonia; Luo, Tao; Gao, Qian; Kato-Maeda, Midori; Ballif, Marie; Egger, Matthias; Macedo, Rita; Mardassi, Helmi; Moreno, Milagros; Vilanova, Griselda Tudo; Fyfe, Janet; Globan, Maria; Thomas, Jackson; Jamieson, Frances; Guthrie, Jennifer L; Asante-Poku, Adwoa; Yeboah-Manu, Dorothy; Wampande, Eddie; Ssengooba, Willy; Joloba, Moses; Boom, W Henry; Basu, Indira; Bower, James; Saraiva, Margarida; Vasconcellos, Sidra E G; Suffys, Philip; Koch, Anastasia; Wilkinson, Robert; Gail-Bekker, Linda; Malla, Bijaya; Ley, Serej D; Beck, Hans-Peter; de Jong, Bouke C; Toit, Kadri; Sanchez-Padilla, Elisabeth; Bonnet, Maryline; Gil-Brusola, Ana; Frank, Matthias; Penlap Beng, Veronique N; Eisenach, Kathleen; Alani, Issam; Ndung'u, Perpetual Wangui; Revathi, Gunturu; Gehre, Florian; Akter, Suriya; Ntoumi, Francine; Stewart-Isherwood, Lynsey; Ntinginya, Nyanda E; Rachow, Andrea; Hoelscher, Michael; Cirillo, Daniela Maria; Skenders, Girts; Hoffner, Sven; Bakonyte, Daiva; Stakenas, Petras; Diel, Roland; Crudu, Valeriu; Moldovan, Olga; Al-Hajoj, Sahal; Otero, Larissa; Barletta, Francesca; Carter, E Jane; Diero, Lameck; Supply, Philip; Comas, Iñaki; Niemann, Stefan; Gagneux, Sebastien

    2016-12-01

    Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

  11. Frequencies of restriction sites.

    PubMed Central

    Waterman, M S

    1983-01-01

    Restriction sites or other sequence patterns are usually assumed to occur according to a Poisson distribution with mean equal to the reciprocal of the probability of the given site or pattern. For situations where non-overlapping occurrences of patterns, such as restriction sites, are the objects of interest, this note shows that the Poisson assumption is frequently misleading. Both the case of base composition (independent bases) and of dinucleotide frequencies (Markov chains) are treated. Moreover, a new technique is presented which allows treatment of collections of patterns, where the departure from the Poisson assumption is even more striking. This later case includes double digests, and an example of a five enzyme digest is included. PMID:6324109

  12. License restrictions at Barnwell

    SciTech Connect

    Autry, V.R.

    1991-12-31

    The State of South Carolina was delegated the authority by the US Nuclear Regulatory Commission to regulate the receipt, possession, use and disposal of radioactive material as an Agreement State. Since 1970, the state has been the principal regulatory authority for the Barnwell Low-Level Waste Disposal Facility operated by Chem-Nuclear Systems, Inc. The radioactive material license issued authorizing the receipt and disposal of low-level waste contains numerous restrictions to ensure environmental protection and compliance with shallow land disposal performance criteria. Low-level waste has evolved from minimally contaminated items to complex waste streams containing high concentrations of radionuclides and processing chemicals which necessitated these restrictions. Additionally, some waste with their specific radionuclides and concentration levels, many classified as low-level radioactive waste, are not appropriate for shallow land disposal unless additional precautions are taken. This paper will represent a number of these restrictions, the rationale for them, and how they are being dealt with at the Barnwell disposal facility.

  13. Revival of the identification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, therapy and vaccine development.

    PubMed

    Liu, Jun; Zhang, Shihong; Tan, Shuguang; Zheng, Beiwen; Gao, George F

    2011-03-01

    Immunogenic T-cell epitopes have a central role in the cellular immunity against pathogens and tumors. However, in the early stage of cellular immunity studies, it was complicated and time-consuming to identify and characterize T-cell epitopes. Currently, the epitope screening is experiencing renewed enthusiasm due to advances in novel techniques and theories. Moreover, the application of T-cell epitope-based diagnoses for tuberculosis and new data on epitope-based vaccine development have also revived the field. There is a growing knowledge on the emphasis of epitope-stimulated T-cell immune responses in the elimination of pathogens and tumors. In this review, we outline the significance of the identification and characterization of T-cell epitopes. We also summarize the methods and strategies for epitope definition and, more importantly, address the relevance of cytotoxic T-lymphocyte epitopes to clinical diagnoses, therapy and vaccine development.

  14. Characterization of a cashew allergen, 11S globulin (Ana o 2), conformational epitope.

    PubMed

    Robotham, Jason M; Xia, Lixin; Willison, LeAnna N; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    Both linear and conformational epitopes likely contribute to the allergenicity of tree nut allergens, yet, due largely to technical issues, few conformational epitopes have been characterized. Using the well studied recombinant cashew allergen, Ana o 2, an 11S globulin or legumin, we identified a murine monoclonal antibody which recognizes a conformational epitope and competes with patient IgE Ana o 2-reactive antibodies. This epitope is expressed on the large subunit of Ana o 2, but only when associated with an 11S globulin small subunit. Both Ana o 2 and the homologous soybean Gly m 6 small subunits can foster epitope expression, even when the natural N-terminal to C-terminal subunit order is reversed in chimeric molecules. The epitope, which is also expressed on native Ana o 2, is readily susceptible to destruction by physical and chemical denaturants.

  15. Tissue invasiveness and non-acidic pH in human candidiasis correlate with “in vivo” expression by Candida albicans of the carbohydrate epitope recognised by new monoclonal antibody 1H4

    PubMed Central

    Monteagudo, C; Viudes, A; Lazzell, A; Martinez, J P; Lopez-Ribot, J L

    2004-01-01

    Background: The morphogenetic conversion between yeast and hyphal growth forms appears to be crucial in the pathogenesis of invasive candidiasis, and can be regulated by environmental signals such as extracellular pH. Aims: To characterise the epitope recognised by monoclonal antibody 1H4, and to evaluate the expression of its corresponding epitope in Candida albicans cells under different conditions of pH and temperature, and “in vivo”, in tissue samples from patients with human candidiasis. Methods: Monoclonal antibody 1H4 was generated against the 58 kDa cell wall mannoprotein of C albicans (mp58), and was further characterised by immunoblot analysis, periodate treatment of the antigenic preparations, and agglutination experiments of C albicans strains 3153A, SC5314, and 412, cultured under different environmental conditions (growth media and pH). An immunohistochemical study was performed in 24 human tissue samples from patients with mucocutaneous and systemic candidiasis. Results: 1H4 recognises a pH sensitive carbohydrate epitope on the surface of C albicans cells, and this epitope is not restricted to mp58, but is shared with other cell wall mannoproteins. Immunohistochemical findings indicated that expression of the 1H4 epitope on C albicans cells in tissue sections from human candidiasis correlates with tissue invasion and pH of the niche. 1H4 immunoreactivity was also found in candida remnants within macrophages. Conclusions: The fact that 1H4 epitope expression selectively identifies invasive forms of C albicans, in addition to candida remnants within macrophages, supports its potential value in the diagnosis and management of human candidiasis. PMID:15166263

  16. Use of recombinant protein to identify a motif-negative human cytotoxic T-cell epitope presented by HLA-A2 in the hepatitis C virus NS3 region.

    PubMed Central

    Kurokohchi, K; Akatsuka, T; Pendleton, C D; Takamizawa, A; Nishioka, M; Battegay, M; Feinstone, S M; Berzofsky, J A

    1996-01-01

    To define cytotoxic T-cell (CTL) epitopes, the common approach involving the use of a series of overlapping synthetic peptides covering the whole protein sequence is impractical for large proteins. Motifs identify only a fraction of epitopes. To identify human CTL epitopes in the NS3 region of hepatitis C virus (HCV), we modified an approach using recombinant protein and the ability of short peptides to bind to class I major histocompatibility complex (MHC) molecules. Peripheral blood mononuclear cells from an HCV-infected patient were stimulated with a proteolytic digest of the recombinant NS3 protein to expand CTL to any active peptides in the digest. The digest was fractionated by reverse-phase high-performance liquid chromatography, and fractions were assessed for the ability to sensitize targets for lysis by CTL. The most active fraction was sequenced, identifying a 15-residue peptide (NS3-1J; TITTGAPVTYSTYGK). This sequence was confirmed to be the source of the activity by synthesis of the corresponding peptide. CTL lines specific for NS3-1J were established from two HCV-infected patients (both HLA-A2 and -B7 positive) by stimulation with the synthetic peptide in vitro. The CTL were HLA-A2 restricted, and the minimal epitope was mapped to a decapeptide NS3-1J (10.4). As this minimal epitope lacks the common HLA-A2-binding motif, this technique is useful for mapping CTL epitopes independent of known motifs and without the requirement for enormous numbers of overlapping peptides. Because this peptide is presented by the most common HLA class I molecule, present in almost half the population, it might be a useful component of a vaccine against HCV. PMID:8523531

  17. Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein.

    PubMed

    Li, F; Riddell, M A; Seow, H F; Takeda, N; Miyamura, T; Anderson, D A

    2000-04-01

    A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats. Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses. Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation. Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 [Li et al. (1997) Journal of Medical Virology 52:289-300], a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients. Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition). Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection.

  18. A recombinant fusion protein displaying murine and human MHC class I- and II-specific epitopes protects against Leishmania amazonensis infection.

    PubMed

    Martins, Vívian T; Lage, Daniela P; Duarte, Mariana C; Carvalho, Ana Maria R S; Costa, Lourena E; Mendes, Tiago A O; Vale, Danniele L; Menezes-Souza, Daniel; Roatt, Bruno M; Tavares, Carlos A P; Soto, Manuel; Coelho, Eduardo A F

    2017-03-01

    Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

  19. Immunogenicity characterization of the multi-epitope vaccine CTB-UE with chitosan-CpG as combination adjuvants against Helicobacter pylori.

    PubMed

    Xing, Yingying; Liu, Wei; Li, Xiaokang; Guo, Le; Lv, Xiaobo; Xi, Tao

    2015-07-03

    Urease is considered as an excellent vaccine candidate antigen against Helicobacter pylori (H. pylori) infection. Our previous study reported a novel multi-epitope vaccine CTB-UE which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and five cell epitopes from urease subunits. Murine experiments indicated that it could induce cellular and humoral immune responses intensively and attenuate H. pylori infection effectively in mice model. However, the body expression and lack of suitable adjuvant of this epitope vaccine restricted its application. In this study, new recombinant Escherichia coli strains was established to increase the solubility by fusing thioredoxin (Trx) and the combination adjuvants which composed of the chitosan and CpG were adopted to enhance the immunogenicity of CTB-UE for oral immunization. The experimental results indicated that the levels of IgG2a, IgG1 and IgA in the serum and the levels of sIgA in stomach, intestine and feces were significantly higher in the vaccinated group compared with the model control group. Additionally, chitosan-CpG combination adjuvants changed the ratio of IgG2a/IgG1 and conferred Th1/Th17-mediated protective immune responses. These results demonstrate that the oral vaccine with chitosan-CpG as combination adjuvants may be a promising vaccine candidate against H. pylori infection.

  20. Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

    PubMed

    Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

    2014-11-04

    Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

  1. A novel multi-epitope peptide vaccine against cancer: an in silico approach.

    PubMed

    Nezafat, Navid; Ghasemi, Younes; Javadi, Gholamreza; Khoshnoud, Mohammad Javad; Omidinia, Eskandar

    2014-05-21

    Cancer immunotherapy has an outstanding position in cancer prevention and treatment. In this kind of therapy, the immune system is activated to eliminate cancerous cells. Multi-epitope peptide cancer vaccines are manifesting as the next generation of cancer immunotherapy. In the present study, we have implemented various strategies to design an efficient multi-epitope vaccine. CD8+ cytolytic T lymphocytes (CTLs) epitopes, which have a pivotal role in cellular immune responses, helper epitopes and adjuvant, are three crucial components of peptide vaccine. CTL epitopes were determined from two high immunogenic protein Wilms tumor-1 (WT1) and human papillomavirus (HPV) E7 by various servers, which apply different algorithms. CTL epitopes were linked together by AAY and HEYGAEALERAG motifs to enhance epitope presentation. Pan HLA DR-binding epitope (PADRE) peptide sequence and helper epitopes, which have defined from Tetanus toxin fragment C (TTFrC) by various servers, were used to induce CD4+ helper T lymphocytes (HTLs) responses. Additionally, helper epitopes were conjugated together via GPGPG motifs that stimulate HTL immunity. Heparin-Binding Hemagglutinin (HBHA), a novel TLR4 agonist was employed as an adjuvant to polarize CD4+ T cells toward T-helper 1 to induce strong CTL responses. Moreover, the EAAAK linker was introduced to N and C terminals of HBHA for efficient separation. 3D model of protein was generated and predicted B cell epitopes were determined from the surface of built structure. Our protein contains several linear and conformational B cell epitopes, which suggests the antibody triggering property of this novel vaccine. Hence, our final protein can be used for prophylactic or therapeutic usages, because it can potentially stimulate both cellular and humoral immune responses.

  2. Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073.

    PubMed

    Fytili, P; Dalekos, G N; Schlaphoff, V; Suneetha, P V; Sarrazin, C; Zauner, W; Zachou, K; Berg, T; Manns, M P; Klade, C S; Cornberg, M; Wedemeyer, H

    2008-07-23

    The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.

  3. The antigen specificity of the rheumatoid arthritis-associated ACPA directed to citrullinated fibrin is very closely restricted.

    PubMed

    Iobagiu, Cristina; Magyar, Anna; Nogueira, Leonor; Cornillet, Martin; Sebbag, Mireille; Arnaud, Jacques; Hudecz, Ferenc; Serre, Guy

    2011-12-01

    The major targets of the disease-specific autoantibodies to citrullinated proteins (ACPA) in synovium of rheumatoid arthritis (RA) patients are borne by the citrullinated α- and β-chains of fibrin. We demonstrated that ACPA target a limited set of citrullinated fibrin peptides and particularly four multicitrullinated peptides which present the major epitopes. In this study, we established the clear immunodominance of the peptides α36-50Cit(38,42) and β60-74Cit(60,72,74) which were recognised by 51/81 (63%) and 61/81 (75%) of ACPA-positive patients, respectively, more than 90% recognising one, the other or both peptides. We also identified the citrullyl residues αCit(42), βCit(72) and βCit(74) as essential for antigenicity, and at a lesser degree αCit(38). Then, we assayed on overlapping 7-mer peptides encompassing the sequences of the two peptides, 3 series of sera recognising either α36-50Cit(38,42) or β60-74Cit(60,72,74) or both peptides. In each series, the reactivity profiles of the sera, largely superimposable, allowed identification of the two 4/5-mer overlapping epitopes (α: VECit(42)HQ and α': Cit(38)VVE), and the single 5-mer epitope (β: GYCit(72)ACit(74)), all located to a flexible globular domain of fibrin on a topological 3D model. In conclusion, we demonstrated that only 3 immunodominant epitopes are targeted by ACPA on citrullinated fibrin stressing their actual oligoclonality. However, the reactivity to the 3 epitopes distinguishes three subgroups of patients. The closely restricted antigen specificity suggests that the autoimmune reaction to citrullinated fibrin is antigen-driven. The accessibility of the epitopes reinforces the hypothesis of a pathogenic role for ACPA via immune complexe formation in the synovial tissue.

  4. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    PubMed

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  5. Machine learning-based methods for prediction of linear B-cell epitopes.

    PubMed

    Wang, Hsin-Wei; Pai, Tun-Wen

    2014-01-01

    B-cell epitope prediction facilitates immunologists in designing peptide-based vaccine, diagnostic test, disease prevention, treatment, and antibody production. In comparison with T-cell epitope prediction, the performance of variable length B-cell epitope prediction is still yet to be satisfied. Fortunately, due to increasingly available verified epitope databases, bioinformaticians could adopt machine learning-based algorithms on all curated data to design an improved prediction tool for biomedical researchers. Here, we have reviewed related epitope prediction papers, especially those for linear B-cell epitope prediction. It should be noticed that a combination of selected propensity scales and statistics of epitope residues with machine learning-based tools formulated a general way for constructing linear B-cell epitope prediction systems. It is also observed from most of the comparison results that the kernel method of support vector machine (SVM) classifier outperformed other machine learning-based approaches. Hence, in this chapter, except reviewing recently published papers, we have introduced the fundamentals of B-cell epitope and SVM techniques. In addition, an example of linear B-cell prediction system based on physicochemical features and amino acid combinations is illustrated in details.

  6. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    PubMed

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  7. Comparative Analysis of Human B Cell Epitopes Based on BCG Genomes

    PubMed Central

    Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

    Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently. Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs, M. bovis AF2122/97, and M. tuberculosis H37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency. Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development. PMID:27382565

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

    PubMed

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

    2013-01-15

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

  9. Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response.

    PubMed

    Zhou, Bin; Liu, Ke; Jiang, Yan; Wei, Jian-Chao; Chen, Pu-Yan

    2011-07-30

    Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV). Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865) and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716), were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine) than that of mono-epitope peptide(rE2-a or rE2-b). Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals) vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

  10. DNA vaccine with discontinuous T-cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi-epitope Mycobacterium tuberculosis vaccine.

    PubMed

    Wu, Manli; Li, Min; Yue, Yan; Xu, Wei

    2016-09-01

    DNA-based vaccine is a promising candidate for immunization and induction of a T-cell-focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi-functional T response against multi-TB antigens, a multi-epitope DNA vaccine and a 'protein backbone grafting' design method is adopted to graft five discontinuous T-cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T-cell epitopes derived from ESAT-6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope-scaffold construct was significantly more potent to induce IFN-γ(+) T response to five T-cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi-functional-CD4(+) T response. It also enhanced granzyme B(+) CTL and IL-2(+) CD8(+) T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi-epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.

  11. Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes.

    PubMed

    Gao, Jun; Gong, Yuping; Zhao, Ping; Zhu, Qing; Yang, Xiaoping; Qi, Zhongtian

    2006-10-01

    Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients' sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.

  12. Maternal smoking, intrauterine growth restriction, and placental apoptosis.

    PubMed

    Vogt Isaksen, Christina

    2004-01-01

    Pregnant women who smoke are at greater risk of delivering a growth-restricted infant than nonsmoking mothers. We wanted to see if apoptosis could be involved in the mechanisms behind smoke-induced growth restriction, and our aim was to compare apoptosis in the placenta of smoking mothers giving birth to growth-restricted infants and nonsmoking mothers with infants of appropriate weight. The project was conducted at the Magee--Womens Hospital and Magee--Womens Research Institute, University of Pittsburgh, PA. Histological sections from 20 placentas were selected from smoking mothers who had given birth to small-for-gestational-age infants (birth weight < or = 2 SD). The controls were gestational-age matched nonsmoking mothers with infants having appropriate-for-gestational-age weight. The TUNEL method was used to demonstrate DNA fragmentation in nuclei, and a monoclonal antibody M30, specific for a neo-epitope on cytokeratin 18, was used to identify apoptotic epithelial cells. The positive nuclei (TUNEL) and positive cells (M30-positive cytoplasm) were counted blindly both in villous tissue and in decidual/basal plate tissue. M30-positive cells in villous tissues were significantly increased in placentas from smoking mothers compared to nonsmoking mothers. When evaluated by the TUNEL method, the difference between the two groups of women was not significant. Our study shows that apoptosis was increased in the placentas of smoking mothers with growth-restricted infants. The difference between the two groups was mainly in the syncytiotrophoblast layer and in connection with perivillous fibrin deposition. Cigarette smoke with reduction in blood flow has previously been shown to increase apoptosis, and it is possible that this could be one of the mechanisms playing a role in the growth restriction.

  13. Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein

    PubMed Central

    Jagessar, S. Anwar; Holtman, Inge R.; Hofman, Sam; Morandi, Elena; Heijmans, Nicole; Laman, Jon D.; Gran, Bruno; Faber, Bart W.; van Kasteren, Sander I.; Eggen, Bart J. L.

    2016-01-01

    EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E–restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20+ spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40–48 core epitope. Finally, LCV infection also induced expression of LC3-II+ cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40–48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40–48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs. PMID:27412414

  14. Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope.

    PubMed

    Chen, Yao-Shen; Chen, Bao-Chen; Lin, You-Sheng; Chang, Jenn-Tzong; Huang, Tsi-Shu; Chen, Jih-Jung; Chang, Tsung-Hsien

    2013-10-01

    Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.

  15. B cell epitope specificity in ANCA-associated vasculitis: does it matter?

    PubMed

    van der Geld, Y M; Stegeman, C A; Kallenberg, C G M

    2004-09-01

    Pauci-immune idiopathic small-vessel vasculitis is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA). Antibodies to PR3 predominate in patients with Wegener's granulomatosis; antibodies to myeloperoxidase (MPO) are found more frequently in patients with microscopic polyangiitis. There is increasing in vivo and in vitro evidence for a pathogenic role of ANCA in systemic vasculitis based on associations of ANCA with disease activity. If ANCA are pathogenic, why is the course of disease different from one patient to another? Antibodies can recognize different binding sites (epitopes) on their corresponding antigens. Differences in binding specificity may influence the pathogenic potential of the antibodies. Differences between epitope specificity of ANCA between patients or changes in epitope specificity of ANCA in time in an individual patient may, accordingly, result in differences in disease expression. This review will focus on epitope specificity of autoantibodies in systemic autoimmune diseases and especially on the epitope specificity of PR3- and MPO-ANCA. We will discuss whether PR3-ANCA or MPO-ANCA recognize different epitopes on PR3 and MPO, respectively, and whether the epitopes recognized by ANCA change in parallel with the disease activity of ANCA-associated vasculitis. Finally, we will speculate if the direct pathogenic role of ANCA can be ascribed to one relapse- or disease-inducing epitope. Characterization of relapse- or disease-inducing epitopes bound by PR3-ANCA and MPO-ANCA is significant for understanding initiation and reactivation of ANCA-associated vasculitis. Elucidating a disease-inducing epitope bound by ANCA may lead to the development of epitope-specific therapeutic strategies.

  16. Identification of linear B-cell epitopes within Tarp of Chlamydia trachomatis.

    PubMed

    Zhu, Shanli; Feng, Yan; Chen, Jun; Lin, Xiaoyun; Xue, Xiangyang; Chen, Shao; Zhong, Xiaozhi; Li, WenShu; Zhang, Lifang

    2014-12-01

    Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin-recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and six possible linear B-cell epitopes peptides (aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152-170 elicited serum immunoglobulin G (IgG) response and epitope 171-186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171-186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171-186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171-186) was an immunodominant B-cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well-documented epitopes, might be included into a candidate epitope-based vaccine against C. trachomatis.

  17. Vaccine Design for H5N1 Based on B- and T-cell Epitope Predictions

    PubMed Central

    Tambunan, Usman Sumo Friend; Sipahutar, Feimmy Ruth Pratiwi; Parikesit, Arli Aditya; Kerami, Djati

    2016-01-01

    From 2003 to 2013, Indonesia had the highest number of avian influenza A cases in humans, with 192 cases and 160 fatalities. Avian influenza is caused by influenza virus type A, such as subtype H5N1. This virus has two glycoproteins: hemagglutinin and neuraminidase, which will become the primary target to be neutralized by vaccine. Vaccine is the most effective immunologic intervention. In this study, we use the epitope-based vaccine design from hemagglutinin and neuraminidase of H5N1 Indonesian strain virus by using immunoinformatics approach in order to predict the binding of B-cell and T-cell epitopes (class I and class II human leukocyte antigen [HLA]). BCPREDS was used to predict the B-cell epitope. Propred, Propred I, netMHCpan, and netMHCIIpan were used to predict the T-cell epitope. Two B-cell epitopes of hemagglutinin candidates and one B-cell epitope of neuraminidase candidates were obtained to bind T-cell CD4+ (class II HLA), and also five T-cell epitope hemagglutinin and four T-cell epitope neuraminidase were obtained to bind T-cell CD8+ (class I HLA). The visualization of epitopes was done using MOE 2008.10. It shows that the binding affinity of epitope–HLA was based on minimum binding free energy (ΔGbinding). Based on this result, visualization, and dynamic simulation, four hemagglutinin epitopes (MEKIVLLLA, CPYLGSPSF, KCQTPMGAI, and IGTSTLNQR) and two neuraminidase epitopes (NPNQKIITI and CYPDAGEIT) were computed as having the best binding affinity from HLA ligand. The results mentioned above are from in silico experiments and need to be validated using wet experiment. PMID:27147821

  18. IMMUNOCAT-a data management system for epitope mapping studies.

    PubMed

    Chung, Jo L; Sun, Jian; Sidney, John; Sette, Alessandro; Peters, Bjoern

    2010-01-01

    To enable rationale vaccine design, studies of molecular and cellular mechanisms of immune recognition need to be linked with clinical studies in humans. A major challenge in conducting such translational research studies lies in the management and integration of large amounts and various types of data collected from multiple sources. For this purpose, we have established "IMMUNOCAT", an interactive data management system for the epitope discovery research projects conducted by our group. The system provides functions to store, query, and analyze clinical and experimental data, enabling efficient, systematic, and integrative data management. We demonstrate how IMMUNOCAT is utilized in a large-scale research contract that aims to identify epitopes in common allergens recognized by T cells from human donors, in order to facilitate the rational design of allergy vaccines. At clinical sites, demographic information and disease history of each enrolled donor are captured, followed by results of an allergen skin test and blood draw. At the laboratory site, T cells derived from blood samples are tested for reactivity against a panel of peptides derived from common human allergens. IMMUNOCAT stores results from these T cell assays along with MHC:peptide binding data, results from RAST tests for antibody titers in donor serum, and the respective donor HLA typing results. Through this system, we are able to perform queries and integrated analyses of the various types of data. This provides a case study for the use of bioinformatics and information management techniques to track and analyze data produced in a translational research study aimed at epitope identification.

  19. Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

    PubMed Central

    Pogue, R R; Eron, J; Frelinger, J A; Matsui, M

    1995-01-01

    Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development. PMID:7545295

  20. Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope.

    PubMed

    Dahan, Rony; Gebe, John A; Preisinger, Anton; James, Eddie A; Tendler, Mark; Nepom, Gerald T; Reiter, Yoram

    2013-12-01

    The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555-567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.

  1. Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope.

    PubMed

    Tan, Amabel C L; Eriksson, Emily M Y; Kedzierska, Katherine; Deliyannis, Georgia; Valkenburg, Sophie A; Zeng, Weiguang; Jackson, David C

    2012-05-01

    In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.

  2. Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding

    NASA Astrophysics Data System (ADS)

    He, Linling; Cheng, Yushao; Kong, Leopold; Azadnia, Parisa; Giang, Erick; Kim, Justin; Wood, Malcolm R.; Wilson, Ian A.; Law, Mansun; Zhu, Jiang

    2015-08-01

    Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

  3. Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes

    PubMed Central

    Abidin, R S; Lua, L H L; Middelberg, A P J; Sainsbury, F

    2015-01-01

    The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles. PMID:26401641

  4. Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors

    PubMed Central

    Bracamontes, John R.; Akk, Gustav; Steinbach, Joe Henry

    2016-01-01

    Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression. PMID:26963253

  5. Bioinformatic screening and detection of allergen cross-reactive IgE-binding epitopes.

    PubMed

    McClain, Scott

    2017-02-13

    Protein allergens can be related by cross-reactivity. Allergens that share relevant sequence can cross-react, those lacking similarity in their IgE antibody-binding epitopes do not cross-react. Cross-reactivity is based on shared epitopes which is due to shared sequence and higher level structure (charge and shape). Epitopes can be important in predicting cross-reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (E-value) was used to identify a threshold specific to identifying cross-reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and Birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross-reactive or other homologous allergens. A FASTA E-value range (1×10(-5) - 1×10(-6) ) was identified that could act as a threshold to help identify cross-reactivity potential. This article is protected by copyright. All rights reserved.

  6. Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses.

    PubMed

    Ripoll, Daniel R; Khavrutskii, Ilja; Wallqvist, Anders; Chaudhury, Sidhartha

    2016-10-18

    Cryo-electron-microscopy (cryo-EM) structures of flaviviruses reveal significant variation in epitope occupancy across different monoclonal antibodies that have largely been attributed to epitope-level differences in conformation or accessibility that affect antibody binding. The consequences of these variations for macroscopic properties such as antibody binding and neutralization are the results of the law of mass action-a stochastic process of innumerable binding and unbinding events between antibodies and the multiple binding sites on the flavivirus in equilibrium-that cannot be directly imputed from structure alone. We carried out coarse-grained spatial stochastic binding simulations for nine flavivirus antibodies with epitopes defined by cryo-EM or x-ray crystallography to assess the role of epitope spatial arrangement on antibody-binding stoichiometry, occupancy, and neutralization. In our simulations, all epitopes were equally competent for binding, representing the upper limit of binding stoichiometry that results from epitope spatial arrangement alone. Surprisingly, our simulations closely reproduced the relative occupancy and binding stoichiometry observed in cryo-EM, without having to account for differences in epitope accessibility or conformation, suggesting that epitope spatial arrangement alone may be sufficient to explain differences in binding occupancy and stoichiometry between antibodies. Furthermore, we found that there was significant heterogeneity in binding configurations even at saturating antibody concentrations, and that bivalent antibody binding may be more common than previously thought. Finally, we propose a structure-based explanation for the stoichiometric threshold model of neutralization.

  7. Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    PubMed

    Palacios-Rodríguez, Yadira; Gazarian, Tatiana; Rowley, Merrill; Majluf-Cruz, Abraham; Gazarian, Karlen

    2007-02-01

    Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

  8. A mimotope gene encoding the major IgE epitope of allergen Phl p 5 for epitope-specific immunization.

    PubMed

    Wallmann, J; Proell, M; Stepanoska, T; Hantusch, B; Pali-Schöll, I; Thalhamer, T; Thalhamer, J; Jensen-Jarolim, E; Hartl, A

    2009-01-29

    A gene vaccine based on a mammalian expression vector containing the sequence of a peptide mimotope of Phl p 5 was constructed. To test whether mimotope gene vaccines can induce allergen-specific antibody responses via molecular mimicry, BALB/c mice were immunized using the mimotope construct with or without a tetanus toxin T-helper epitope. Moreover, intradermal injection was compared to epidermal application via gene gun immunization. Immunization with both mimotope gene constructs elicited allergen-specific antibody responses. As expected, gene gun bombardment induced a Th2-biased immune response, typically associated with IgG1 and IgE antibody production. In contrast, intradermal injection of the vaccine triggered IgG2a antibody expression without any detectable IgE levels, thus biasing the immune response towards Th1. In an RBL assay, mimotope-specific IgG antibodies were able to prevent cross-linking of allergen-specific IgE by Phl p 5. A construct coding for the complete Phl p 5 induced T-cell activation, IFN-gamma and IL-4 production. In contrast, the mimotope-DNA construct being devoid of allergen-specific T-cell epitopes had no capacity to activate allergen-specific T cells. Taken together, our data show that it is feasible to induce blocking IgG antibodies with a mimotope-DNA construct when applied intradermally. Thus the mimotope-DNA strategy has two advantages: (1) the avoidance of IgE induction and (2) the avoidance of triggering allergen-specific T-lymphocytes. We therefore suggest that mimotope gene vaccines are potential candidates for epitope-specific immunotherapy of type I allergy.

  9. Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking

    PubMed Central

    Bergström, Joakim J. E.; Xu, Hui; Heyman, Birgitta

    2017-01-01

    Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG–antigen complexes and/or that IgG “hides” the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking. PMID:28321225

  10. Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes.

    PubMed

    Kim, J J; Mandrell, R E; Griffiss, J M

    1989-02-01

    Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

  11. Restrictive vs. non-restrictive composition: a magnetoencephalography study

    PubMed Central

    Leffel, Timothy; Lauter, Miriam; Westerlund, Masha; Pylkkänen, Liina

    2014-01-01

    Recent research on the brain mechanisms underlying language processing has implicated the left anterior temporal lobe (LATL) as a central region for the composition of simple phrases. Because these studies typically present their critical stimuli without contextual information, the sensitivity of LATL responses to contextual factors is unknown. In this magnetoencephalography (MEG) study, we employed a simple question-answer paradigm to manipulate whether a prenominal adjective or determiner is interpreted restrictively, i.e., as limiting the set of entities under discussion. Our results show that the LATL is sensitive to restriction, with restrictive composition eliciting higher responses than non-restrictive composition. However, this effect was only observed when the restricting element was a determiner, adjectival stimuli showing the opposite pattern, which we hypothesise to be driven by the special pragmatic properties of non-restrictive adjectives. Overall, our results demonstrate a robust sensitivity of the LATL to high level contextual and potentially also pragmatic factors. PMID:25379512

  12. Dietary restriction with and without caloric restriction for healthy aging

    PubMed Central

    Lee, Changhan; Longo, Valter

    2016-01-01

    Caloric restriction is the most effective and reproducible dietary intervention known to regulate aging and increase the healthy lifespan in various model organisms, ranging from the unicellular yeast to worms, flies, rodents, and primates. However, caloric restriction, which in most cases entails a 20–40% reduction of food consumption relative to normal intake, is a severe intervention that results in both beneficial and detrimental effects. Specific types of chronic, intermittent, or periodic dietary restrictions without chronic caloric restriction have instead the potential to provide a significant healthspan increase while minimizing adverse effects. Improved periodic or targeted dietary restriction regimens that uncouple the challenge of food deprivation from the beneficial effects will allow a safe intervention feasible for a major portion of the population. Here we focus on healthspan interventions that are not chronic or do not require calorie restriction. PMID:26918181

  13. Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies.

    PubMed Central

    Han, Y; Kanbe, T; Cherniak, R; Cutler, J E

    1997-01-01

    We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived

  14. Vaccine focusing to cross-subtype HIV-1 gp120 variable loop epitopes.

    PubMed

    Cardozo, Timothy; Wang, Shixia; Jiang, Xunqing; Kong, Xiang-Peng; Hioe, Catarina; Krachmarov, Chavdar

    2014-09-03

    We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross-subtype HIV-1 neutralizing Abs can be intentionally elicited in mammals by vaccination. The precise boundaries of the epitopes and conformational flexibility in the presentation of the epitopes in the immunogen appeared to be important for successful elicitation. This work may serve as a starting point for translating the activities of human broadly neutralizing anti-HIV-1 monoclonal antibodies (bNAbs) into matched immunogens that can contribute to an efficacious HIV-1 vaccine.

  15. Protein grafting of an HIV-1-inhibiting epitope

    NASA Astrophysics Data System (ADS)

    Sia, Samuel K.; Kim, Peter S.

    2003-08-01

    Protein grafting, the transfer of a binding epitope of one ligand onto the surface of another protein, is a potentially powerful technique for presenting peptides in preformed and active three-dimensional conformations. Its utility, however, has been limited by low biological activity of the designed ligands and low tolerance of the protein scaffolds to surface substitutions. Here, we graft the complete binding epitope (19 nonconsecutive amino acids with a solvent-accessible surface area of >2,000 Å2) of an HIV-1 C-peptide, which is derived from the C-terminal region of HIV-1 gp41 and potently inhibits HIV-1 entry into cells, onto the surface of a GCN4 leucine zipper. The designed peptide, named C34coil, displays a potent antiviral activity approaching that of the native ligand. Moreover, whereas the linear C-peptide is unstructured and sensitive to degradation by proteases, C34coil is well structured, conformationally stable, and exhibits increased resistance to proteolytic degradation compared with the linear peptide. In addition to being a structured antiviral inhibitor, C34coil may also serve as the basis for the development of an alternative class of immunogens. This study demonstrates that "one-shot" protein grafting, without subsequent rounds of optimization, can be used to create ligands with structural conformations and improved biomedical properties.

  16. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    SciTech Connect

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  17. Improving wheat to remove coeliac epitopes but retain functionality.

    PubMed

    Shewry, Peter R; Tatham, Arthur S

    2016-01-01

    Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a "coeliac-safe" wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research.

  18. Improving wheat to remove coeliac epitopes but retain functionality

    PubMed Central

    Shewry, Peter R.; Tatham, Arthur S.

    2016-01-01

    Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a “coeliac-safe” wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research. PMID:26937068

  19. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  20. Conservation of HIV-1 T cell epitopes across time and clades: validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine.

    PubMed

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou; Ardito, Matthew T; Boyle, Christine M; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D; De Groot, Anne S

    2012-12-14

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine.

  1. Conservation of HIV-1 T cell epitopes across time and clades: Validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine

    PubMed Central

    Levitz, Lauren; Koita, Ousmane A.; Sangare, Kotou; Ardito, Matthew T.; Boyle, Christine M.; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M.; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D.; De Groot, Anne S.

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the “Achilles’ heel” of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. PMID:23102976

  2. Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

    SciTech Connect

    Yu Hua; Jiang Lifang . E-mail: jianglf909@yahoo.com.cn; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-03-15

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

  3. First report on the antibody verification of HLA-DR, HLA-DQ and HLA-DP epitopes recorded in the HLA Epitope Registry.

    PubMed

    Duquesnoy, Rene J; Marrari, Marilyn; Tambur, Anat R; Mulder, Arend; Sousa, Luiz Cláudio Demes da Mata; da Silva, Adalberto Socorro; do Monte, Semiramis J H

    2014-11-01

    The International Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to HLA mismatches. These epitopes can be structurally defined as eplets by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. A major goal is to identify HLA eplets that have been verified experimentally with informative antibodies. This report addresses class II epitopes encoded by genes in the HLA-D region. Our analysis included reviews of many publications about epitope specificity of class II reactive human and murine monoclonal antibodies and informative alloantibodies from HLA sensitized patients as well as our own antibody testing results. As of July 1, 2014, 24 HLA-DRB1/3/4/5, 15 DQB, 3 DQA and 8 DPB antibody-verified epitopes have been identified and recorded. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

  4. Structural basis for epitope sharing between group 1 allergens of cedar pollen

    PubMed Central

    Midoro-Horiuti, Terumi; Schein, Catherine H.; Mathura, Venkatarajan; Braun, Werner; Czerwinski, Edmund W.; Togawa, Akihisa; Kondo, Yasuto; Oka, Tetsuo; Watanabe, Masanao; Goldblum, Randall M.

    2008-01-01

    The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved β-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development. PMID:15975657

  5. Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA).

    PubMed

    O'Hara, Joanne M; Kasten-Jolly, Jane C; Reynolds, Claire E; Mantis, Nicholas J

    2014-01-01

    Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design.

  6. HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells.

    PubMed

    Kwok, W W; Liu, A W; Novak, E J; Gebe, J A; Ettinger, R A; Nepom, G T; Reymond, S N; Koelle, D M

    2000-04-15

    Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.

  7. The use of multiple-pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti-(recombinant bovine growth hormone) sera. Comparison with predicted regions of immunogenicity and location within the three-dimensional structure of the molecule.

    PubMed

    Beattie, J; Fawcett, H A; Flint, D J

    1992-11-15

    A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth-hormone (rbGH) molecule recognised by three polyclonal guinea-pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea-pig antisera, 3 or 4, very-well-defined major continuous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea-pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring alpha-helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea-pig antiserum. Comparison of the four guinea-pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson-Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods. In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous

  8. Identification and Localization of Minimal MHC-restricted CD8+ T Cell Epitopes within the Plasmodium falciparum AMA1 Protein

    DTIC Science & Technology

    2010-08-24

    Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the...A, Muratova O, Awkal M, et al: Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria...PfCP-2.9, an asexual blood-stage vaccine candidate of Plasmodium falciparum. Malar J 2010, 9(1):94. 40. Senger T, Becker MR, Schadlich L, Waterboer T

  9. Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

    SciTech Connect

    Huang, Pi H.; Li, Ying J.; Su, Yu P.; Lee, Long H.; Liu, Hung J. . E-mail: hjliu@mail.npust.edu.tw

    2005-02-20

    We have previously shown that avian reovirus (ARV) {sigma}A and {sigma}NS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on {sigma}A (I and II) and three epitopes (A, B, and C) on {sigma}NS. To further define the location of epitopes on {sigma}A and {sigma}NS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of {sigma}A and {sigma}NS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on {sigma}A was located at amino acid residues {sup 340}QWVMAGLVSAA{sup 350} and epitope B on {sigma}NS at amino acid residues {sup 180}MLDMVDGRP{sup 188}. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of {sigma}A and {sigma}NS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete {sigma}NS but not with truncated {sigma}NS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on {sigma}NS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on {sigma}A was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the {sigma}NS with MAb 1F9. The {sigma}NS of ARV with ss

  10. Monoclonal antibody epitope mapping of Plasmodium falciparum rhoptry proteins.

    PubMed

    Sam-Yellowe, T Y; Ndengele, M M

    1993-02-01

    Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA2 from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 110-kDa protein. Furthermore, PLA2 activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA2 is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [3H] palmitate and [3H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [35S] methionine-labeled proteins were labeled with [3H]palmitate in ring-infected erythrocyte membranes

  11. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer

    PubMed Central

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer. PMID:25483665

  12. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer.

    PubMed

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.

  13. PepMapper: A Collaborative Web Tool for Mapping Epitopes from Affinity-Selected Peptides

    PubMed Central

    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/ PMID:22701536

  14. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins.

    PubMed

    Sormanni, Pietro; Aprile, Francesco A; Vendruscolo, Michele

    2015-08-11

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

  15. TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes

    PubMed Central

    Chowell, Diego; Krishna, Sri; Cocita, Clément; Shu, Jack; Tan, Xuefang; Greenberg, Philip D.; Klavinskis, Linda S.; Blattman, Joseph N.; Anderson, Karen S.

    2015-01-01

    Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8+ T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development. PMID:25831525

  16. Conservancy of mAb Epitopes in Ebolavirus Glycoproteins of Previous and 2014 Outbreaks

    PubMed Central

    Ponomarenko, Julia; Vaughan, Kerrie; Sette, Alessandro; Maurer-Stroh, Sebastian

    2014-01-01

    Background: Several monoclonal antibodies (mAb) are being evaluated as treatment options for the current 2014 Ebola outbreak. But they were derived from and tested for protection against the older 1976 Mayinga or 1995 Kikwit Zaire Ebolaviruses (EBOV). The EBOV sequences reported for the current outbreak contain several mutations whose significance remained to be established. Methods: We analyzed sequence and structural conservation of the Ebolavirus glycoprotein (GP) epitopes for all experimentally identified protective mAbs published to date. Results: The conservancy analysis of protective mAb epitopes in the Ebolavirus glycoprotein sequences spanning all Ebola virus lineages since 1976 showed that conservancy within the Zaire EBOV lineage was high, with only one immunodominant epitope of mAb 13F6-1-2 acquiring two novel mutations in the 2014 outbreak that might potentially change the antibody specificity and neutralization activity. However, the conservation to other Ebola viruses was unexpectedly low. Conclusion: Low conservancy of Zaire EBOV mAb epitopes to other EBOV lineages suggests that these epitopes are not indispensable for viral fitness, and that alternative mAbs could be developed to broadly target all EBOV. However, average percent sequence identity of the epitopes for mAbs used in current cocktails to the Zaire EBOV is high with only one epitope differing in the 2014 outbreak. These data bode well for general usefulness of these antibodies in the context of the current outbreak. PMID:25642381

  17. Overlap of the cancer genome atlas and the immune epitope database.

    PubMed

    Sait, Shaimaa; Fawcett, Timothy; Blanck, George

    2016-10-01

    Mutant peptides resulting from cancer drivers or passenger mutations are expected to have the potential to serve as a basis for cancer vaccines. However, a number of parameters regulate vaccine-associated immunogenicity, including the suitability of a peptide for binding to an antigen-presenting molecule or antibody. In order to obtain a basic indication of the prospect of human cancer epitope identification via current database development strategies, an overlap of the mutant Homo sapiens epitopes listed on the Immune Epitope Database (IEDB) and the mutant peptides indicated by The Cancer Genome Atlas (TCGA) somatic mutation database was obtained. No putative TCGA mutant peptides were detected among the 8,890 14-18 amino acid (AA) IEDB peptides available. In total, 3 IEDB mutant epitopes that encompassed a TCGA mutant AA position, but did not overlap the exact position of the TCGA mutant AA, were detected. The results of the present analysis confirm that verification of certain aspects of cancer epitope function can be obtained via the continued and systematic expansion of databases representing human protein epitopes. However, the analysis also indicates that there is relatively limited systematic information available regarding antigen-presenting molecule epitopes and cancer-related mutant peptides.

  18. Comparison of coat protein epitopes of two zucchini yellow mosaic virus isolates.

    PubMed

    Kundu, A K; Ohshima, K; Sako, N

    1998-06-01

    Serological differences between two zucchini yellow mosaic virus (ZYMV) isolates (ZYMV-169 and ZYMV-M) obtained from two distinct geographical locations in Japan were determined by mapping epitopes on the coat proteins (CPs) of the two isolates. A total of 45 monoclonal antibodies (MAbs) against the two isolates were produced and the epitopes on the CPs were delineated by reacting these MAbs with trypsin-treated ZYMV particles and Escherichia coli-expressed ZYMV CP fragments. Six MAbs of groups I-a and I-b, specific for ZYMV-169, recognised two epitopes in the N-terminal region at amino acids (aa) 1-28 and 6-41 of ZYMV-169 CP. Fourteen MAbs of group II, specific for ZYMV-M, recognised epitopes in the N-terminal region of ZYMV-M CP. Twenty-one MAbs of groups III-a, III-b(i), III-b(ii), and III-b(iii), reacting with both isolates, recognised four epitopes; one epitope was located in the N-terminal region at aa 6-28 and the remaining three epitopes were located in the core region at aa 42-95, 171-227 and 228-259 of ZYMV CPs.

  19. Multi-epitope schistosome vaccine candidates tested for protective immunogenicity in mice.

    PubMed

    Yang, W; Jackson, D C; Zeng, Q; McManus, D P

    2000-08-15

    The major challenge in the development of anti-schistosome vaccines is to use defined antigens to stimulate an appropriate immune response that leads to resistance. Several promising candidate vaccine antigens including the glycolytic enzyme triose-phosphate isomerase (SmTPI), a 28 kDa glutathione-S-transferase (Sm28), the myofibrilar protein paramyosin (Sm97), an integral membrane protein (Sm23) and calpain (Smcalpain) have been characterised and their primary sequences derived for Schistosoma mansoni. Furthermore, sequences are available for synthetic peptides mimicking epitopes on these molecules capable of inducing schistosome-specific T- and B-cell responses. These schistosome vaccine candidates have generally been tested with varying degrees of success as single components, with only one report of the use of a multivalent antigen or multi-epitope approach. We describe the assembly of multiple defined and different epitopes of S. mansoni into a variety of single covalent structures; these included a DNA vaccine encoding different epitopes in tandem, the polyprotein itself that is encoded by this DNA and branched synthetic peptide epitope-based polymers in which the individual epitopes are pendant from an inert backbone. Each of the vaccine constructs examined, with the exception of the DNA vaccine, generated antibodies that were capable of binding to a tandem sequence of the epitopes. Although these results were encouraging, none of the constructs protected animals from subsequent challenge infection, indicating that the immune responses elicited were inadequate or inappropriate for parasite killing in vivo.

  20. Overlap of the cancer genome atlas and the immune epitope database

    PubMed Central

    Sait, Shaimaa; Fawcett, Timothy; Blanck, George

    2016-01-01

    Mutant peptides resulting from cancer drivers or passenger mutations are expected to have the potential to serve as a basis for cancer vaccines. However, a number of parameters regulate vaccine-associated immunogenicity, including the suitability of a peptide for binding to an antigen-presenting molecule or antibody. In order to obtain a basic indication of the prospect of human cancer epitope identification via current database development strategies, an overlap of the mutant Homo sapiens epitopes listed on the Immune Epitope Database (IEDB) and the mutant peptides indicated by The Cancer Genome Atlas (TCGA) somatic mutation database was obtained. No putative TCGA mutant peptides were detected among the 8,890 14–18 amino acid (AA) IEDB peptides available. In total, 3 IEDB mutant epitopes that encompassed a TCGA mutant AA position, but did not overlap the exact position of the TCGA mutant AA, were detected. The results of the present analysis confirm that verification of certain aspects of cancer epitope function can be obtained via the continued and systematic expansion of databases representing human protein epitopes. However, the analysis also indicates that there is relatively limited systematic information available regarding antigen-presenting molecule epitopes and cancer-related mutant peptides. PMID:27703532

  1. Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza

    PubMed Central

    Zarnitsyna, Veronika I.; Ellebedy, Ali H.; Davis, Carl; Jacob, Joshy; Ahmed, Rafi; Antia, Rustom

    2015-01-01

    The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza's main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains. PMID:26194761

  2. Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks.

    PubMed

    Li, Chenxi; Liu, Junyan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Qingshan; Hua, Ronghong; Liu, Jyung-Hurng; Liu, Ming; Zhang, Yun

    2016-11-10

    Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to (221)LD/NLPW(225) and (87)YAEYI(91) by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas (221)LD/NLPW(225) was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.

  3. In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model

    PubMed Central

    Gutiérrez, Andres H.; Loving, Crystal; Moise, Leonard; Terry, Frances E.; Brockmeier, Susan L.; Hughes, Holly R.; Martin, William D.; De Groot, Anne S.

    2016-01-01

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs. PMID:27411061

  4. Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes

    PubMed Central

    Lindestam Arlehamn, Cecilia S.; Paul, Sinu; Mele, Federico; Huang, Charlie; Greenbaum, Jason A.; Vita, Randi; Sidney, John; Peters, Bjoern; Sallusto, Federica; Sette, Alessandro

    2015-01-01

    A previous unbiased genome-wide analysis of CD4 Mycobacterium tuberculosis (MTB) recognition using peripheral blood mononuclear cells from individuals with latent MTB infection (LTBI) or nonexposed healthy controls (HCs) revealed that certain MTB sequences were unexpectedly recognized by HCs. In the present study, it was found that, based on their pattern of reactivity, epitopes could be divided into LTBI-specific, mixed reactivity, and HC-specific categories. This pattern corresponded to sequence conservation in nontuberculous mycobacteria (NTMs), suggesting environmental exposure as an underlying cause of differential reactivity. LTBI-specific epitopes were found to be hyperconserved, as previously reported, whereas the opposite was true for NTM conserved epitopes, suggesting that intragenus conservation also influences host pathogen adaptation. The biological relevance of this observation was demonstrated further by several observations. First, the T cells elicited by MTB/NTM cross-reactive epitopes in HCs were found mainly in a CCR6+CXCR3+ memory subset, similar to findings in LTBI individuals. Thus, both MTB and NTM appear to elicit a phenotypically similar T-cell response. Second, T cells reactive to MTB/NTM-conserved epitopes responded to naturally processed epitopes from MTB and NTMs, whereas T cells reactive to MTB-specific epitopes responded only to MTB. Third, cross-reactivity could be translated to antigen recognition. Several MTB candidate vaccine antigens were cross-reactive, but others were MTB-specific. Finally, NTM-specific epitopes that elicit T cells that recognize NTMs but not MTB were identified. These epitopes can be used to characterize T-cell responses to NTMs, eliminating the confounding factor of MTB cross-recognition and providing insights into vaccine design and evaluation. PMID:25548174

  5. Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks

    PubMed Central

    Li, Chenxi; Liu, Junyan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Qingshan; Hua, Ronghong; Liu, Jyung-Hurng; Liu, Ming; Zhang, Yun

    2016-01-01

    Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to 221LD/NLPW225 and 87YAEYI91 by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas 221LD/NLPW225 was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV. PMID:27834908

  6. Contribution of Disulfide Bridging to Epitope Expression of the Dengue Type 2 Virus Envelope Glycoprotein

    PubMed Central

    Roehrig, John T.; Volpe, Katharine E.; Squires, Jennifer; Hunt, Ann R.; Davis, Brent S.; Chang, Gwong-Jen J.

    2004-01-01

    The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK2 cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication. PMID:14963174

  7. Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.

    PubMed

    Sharon, Jacqueline; Rynkiewicz, Michael J; Lu, Zhaohua; Yang, Chiou-Ying

    2014-05-01

    Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B-cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding can be localized to specific regions of antigens by immunochemical assays. The epitopes are then identified with mapping methods such as X-ray crystallography of antigen-antibody complexes, antibody inhibition of hydrogen-deuterium exchange in the antigen, antibody-induced alteration of the nuclear magnetic resonance spectrum of the antigen, and experimentally validated computational docking of antigen-antibody complexes. The diversity in shape, size and structure of protective B-cell epitopes, and the increasing importance of protective B-cell epitope discovery to development of vaccines and antibody therapeutics are illustrated through examples from different microbe categories, with emphasis on epitopes targeted by broadly neutralizing antibodies to pathogens of high antigenic variation. Examples include the V-shaped Ab52 glycan epitope in the O-antigen of Francisella tularensis, the concave CR6261 peptidic epitope in the haemagglutinin stem of influenza virus H1N1, and the convex/concave PG16 glycopeptidic epitope in the gp120 V1/V2 loop of HIV type 1.

  8. 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.

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

    PubMed

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

    2016-06-01

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

  10. Mapping epitopes and antigenicity by site-directed masking

    PubMed Central

    Paus, Didrik; Winter, Greg

    2006-01-01

    Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (β-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to β-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with β-lactamase in Freund’s adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund’s adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. PMID:16754878

  11. Characterization of CTL Recognized Epitopes on Human Breast Tumors

    DTIC Science & Technology

    1996-09-01

    restricted cytolytic T lymphocytes. J. Exp. Med. 184:121-130, 1996. 22. Weisberg, J. H., Curcio, M., Caron, P. C., Raghu, G., Mechetner, E. B., Roepe, P...liquid chromatography (HPLC). The MOvl8/MOvl9 monoclonal antibody (mAb) and purity of the peptides used in this study ranged be- appears to be restricted ...to ovarian cancer (10). Be- tween 92 and 95%. The purity and amino acid com- cause of its restricted expression, FBP is also ex- position of these

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

    PubMed

    Chen, Xueni; Dreskin, Stephen C

    2016-12-20

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

  13. Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J.

    PubMed

    Hou, Minbo; Zhou, Defang; Li, Gen; Guo, Huijun; Liu, Jianzhu; Wang, Guihua; Zheng, Qiankun; Cheng, Ziqiang

    2016-03-08

    Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry.

  14. Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

    PubMed

    Robijn, M L M; Wuhrer, M; Kornelis, D; Deelder, A M; Geyer, R; Hokke, C H

    2005-01-01

    The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

  15. Peptide Conformations for a Microarray Surface-Tethered Epitope of the Tumor Suppressor p53

    SciTech Connect

    Feng, Jun; Wong, Ka-Yiu; Lynch, Gillian C.; Gao, Xiaolian; Pettitt, Bernard M.

    2007-12-13

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Peptides or proteins near surfaces exhibit different structural properties from those present in a homogeneous solution, and these differences give rise to varied biological activity. Therefore, understanding the detailed molecular structure of these molecules tethered to a surface is important for interpreting the performance of the various microarrays based on the activities of the immobilized peptides or proteins. We performed molecular dynamics simulations of a pentapeptide, RHSVV, an epitope of the tumor suppressor protein p53, tethered via a spacer on a functionalized silica surface and free in solution, to study their structural and conformational differences. These calculations allowed analyses of the peptide-surface interactions, the sequence orientations, and the translational motions of the peptide on the surface to be performed. Conformational similarities are found among dominant structures of the tethered and free peptide. In the peptide microarray simulations, the peptide fluctuates between a parallel and tilted orientation driven in part by the hydrophobic interactions between the nonpolar peptide residues and the methyl-terminated silica surface. The perpendicular movement of the peptide relative to the surface is also restricted due to the hydrophobic nature of the microarray surface. With regard to structures available for recognition and binding, we find that similar conformations to those found in solution are available to the peptide tethered to the surface, but with a shifted equilibrium constant. Comparisons with experimental results show important implications of this for peptide microarray design and assays.

  16. Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution.

    PubMed

    Neumann-Haefelin, Christoph; McKiernan, Susan; Ward, Scott; Viazov, Sergei; Spangenberg, Hans Christian; Killinger, Thomas; Baumert, Thomas F; Nazarova, Natalja; Sheridan, Isabelle; Pybus, Oliver; von Weizsäcker, Fritz; Roggendorf, Michael; Kelleher, Dermot; Klenerman, Paul; Blum, Hubert E; Thimme, Robert

    2006-03-01

    Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.

  17. Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

    PubMed

    Xi, Jun; Yan, Huili

    2016-10-01

    Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

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

    SciTech Connect

    Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.; Hughes, Colette M.; Hammond, Katherine B.; Ventura, Abigail B.; Reed, Jason S.; Gilbride, Roxanne M.; Ainslie, Emily; Morrow, David W.; Ford, Julia C.; Selseth, Andrea N.; Pathak, Reesab; Malouli, Daniel; Legasse, Alfred W.; Axthelm, Michael K.; Nelson, Jay A.; Gillespie, Geraldine M.; Walters, Lucy C.; Brackenridge, Simon; Sharpe, Hannah R.; Lopez, Cesar Augusto; Fruh, Klaus; Korber, Bette Tina; McMichael, Andrew J.; Gnanakaran, Sandrasegaram; Sacha, Jonah B.; Picker, Louis J.

    2016-02-12

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

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

    DOE PAGES

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

    2016-02-12

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

  20. An immunodominant class I-restricted cytotoxic T lymphocyte determinant of human immunodeficiency virus type 1 induces CD4 class II-restricted help for itself

    PubMed Central

    1990-01-01

    We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC- restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype. PMID:1689366

  1. Analysis of Conformational B-Cell Epitopes in the Antibody-Antigen Complex Using the Depth Function and the Convex Hull.

    PubMed

    Zheng, Wei; Ruan, Jishou; Hu, Gang; Wang, Kui; Hanlon, Michelle; Gao, Jianzhao

    2015-01-01

    The prediction of conformational b-cell epitopes plays an important role in immunoinformatics. Several computational methods are proposed on the basis of discrimination determined by the solvent-accessible surface between epitopes and non-epitopes, but the performance of existing methods is far from satisfying. In this paper, depth functions and the k-th surface convex hull are used to analyze epitopes and exposed non-epitopes. On each layer of the protein, we compute relative solvent accessibility and four different types of depth functions, i.e., Chakravarty depth, DPX, half-sphere exposure and half space depth, to analyze the location of epitopes on different layers of the proteins. We found that conformational b-cell epitopes are rich in charged residues Asp, Glu, Lys, Arg, His; aliphatic residues Gly, Pro; non-charged residues Asn, Gln; and aromatic residue Tyr. Conformational b-cell epitopes are rich in coils. Conservation of epitopes is not significantly lower than that of exposed non-epitopes. The average depths (obtained by four methods) for epitopes are significantly lower than that of non-epitopes on the surface using the Wilcoxon rank sum test. Epitopes are more likely to be located in the outer layer of the convex hull of a protein. On the benchmark dataset, the cumulate 10th convex hull covers 84.6% of exposed residues on the protein surface area, and nearly 95% of epitope sites. These findings may be helpful in building a predictor for epitopes.

  2. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    PubMed

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  3. Characterization of epitopes recognized by monoclonal antibodies: experimental approaches supported by freely accessible bioinformatic tools.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2013-05-01

    Monoclonal antibodies (mAbs) have been used successfully both in research and for clinical purposes. The possible use of protective mAbs directed against different microbial pathogens is currently being considered. The fine definition of the epitope recognized by a protective mAb is an important aspect to be considered for possible development in epitope-based vaccinology. The most accurate approach to this is the X-ray resolution of mAb/antigen crystal complex. Unfortunately, this approach is not always feasible. Under this perspective, several surrogate epitope mapping strategies based on the use of bioinformatics have been developed. In this article, we review the most common, freely accessible, bioinformatic tools used for epitope characterization and provide some basic examples of molecular visualization, editing and computational analysis.

  4. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies.

    PubMed

    Dong, Shiwei; Zhao, Qin; Lu, Mingzhe; Sun, Peiming; Qiu, Hongkai; Zhang, Lu; Lv, Junhua; Zhou, En-Min

    2011-02-01

    Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.

  5. Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

    PubMed Central

    Ferguson, Andrew L.; Falkowska, Emilia; Walker, Laura M.; Seaman, Michael S.; Burton, Dennis R.; Chakraborty, Arup K.

    2013-01-01

    Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens. PMID:24312481

  6. Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

    PubMed Central

    Lesénéchal, Mylène; Becquart, Laurence; Lacoux, Xavier; Ladavière, Laurent; Baida, Renata C. P.; Paranhos-Baccalà, Glaucia; da Silveira, José Franco

    2005-01-01

    Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules. PMID:15699429

  7. Mapping of the immunodominant T cell epitopes of the protein topoisomerase I

    PubMed Central

    Veeraraghavan, S; Renzoni, E; Jeal, H; Jones, M; Hammer, J; Wells, A; Black, C; Welsh, K; du Bois, R M

    2004-01-01

    Objectives: To identify the immunodominant T cell epitopes of the topoisomerase I protein in patients with systemic sclerosis (SSc) and control subjects, using computational analysis software (TEPITOPE) and T cell proliferation assays. Methods: Six oligopeptides, predicted by TEPITOPE software as potential topoisomerase protein epitopes, were used to perform T cell proliferation assays in 21 patients with SSc and 15 healthy controls. Results: A positive response to at least one of the peptides was seen in 10/21 patients and 7/15 healthy controls. Among responders, the proliferative response was limited to a single peptide in 6/7 healthy controls, whereas 5/10 patients responded to more than one peptide. In responding patients a significant correlation was found between disease duration and number of peptides inducing a response (p = 0.007). Conclusions: Several T cell epitopes of the topoisomerase I protein have been identified and evidence has been found to suggest epitope spreading in patients with SSc. PMID:15249326

  8. Epitope expression in nine commercial kits for the determination of anti-thyroid peroxidase (TPO) antibodies.

    PubMed

    Whitham, K; Patel, D; Ward, A M

    1999-01-01

    Anti-thyroid peroxidase (TPO) antibodies, from patients with autoimmune disease, bind predominantly to two neighbouring, non-identical, conformational domains referred to as domains A and B. In recent years a number of ELISA assays have been developed for the detection of anti-TPO antibodies, however, considerable variation between the different commercial assay kits has been documented in inter-laboratory surveys (UK NEQAS). This investigation assessed the differences between nine commercial ELISA assays currently available in the UK. The anti-TPO kits varied in terms of their imprecision and accuracy and in the density of coated antigen. Recombinant antigen containing kits demonstrated partial destruction of the B epitope, possibly due to the close proximity of both epitope regions in the recombinant molecule. None of the kits expressed only one epitope although there were differences in the degrees of expression of each epitope. Clinicians should be aware of the variability of the numbers generated, when interpreting test results.

  9. Longitudinal epitope mapping in MuSK myasthenia gravis: implications for disease severity.

    PubMed

    Huijbers, Maartje G; Vink, Anna-Fleur D; Niks, Erik H; Westhuis, Ruben H; van Zwet, Erik W; de Meel, Robert H; Rojas-García, Ricardo; Díaz-Manera, Jordi; Kuks, Jan B; Klooster, Rinse; Straasheijm, Kirsten; Evoli, Amelia; Illa, Isabel; van der Maarel, Silvère M; Verschuuren, Jan J

    2016-02-15

    Muscle weakness in MuSK myasthenia gravis (MG) is caused predominantly by IgG4 antibodies which block MuSK signalling and destabilize neuromuscular junctions. We determined whether the binding pattern of MuSK IgG4 antibodies change throughout the disease course ("epitope spreading"), and affect disease severity or treatment responsiveness. We mapped the MuSK epitopes of 255 longitudinal serum samples of 53 unique MuSK MG patients from three independent cohorts with ELISA. Antibodies against the MuSK Iglike-1 domain determine disease severity. Epitope spreading outside this domain did not contribute to disease severity nor to pyridostigmine responsiveness. This provides a rationale for epitope specific treatment strategies.

  10. Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

    PubMed

    Angström, J; Teneberg, S; Karlsson, K A

    1994-12-06

    Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

  11. Functional HLA-DR T cell epitopes of CEA identified in patients with colorectal carcinoma immunized with the recombinant protein CEA.

    PubMed

    Ullenhag, Gustav J; Fagerberg, Jan; Strigård, Karin; Frödin, Jan-Erik; Mellstedt, Håkan

    2004-04-01

    A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6-19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine.

  12. Structures of HLA-A*1101 complexed with immunodominant nonamer and decamer HIV-1 epitopes clearly reveal the presence of a middle, secondary anchor residue.

    PubMed

    Li, Lenong; Bouvier, Marlene

    2004-05-15

    HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313-321) (AIFQSSMTK) and decamer Nef(73-82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.

  13. Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model.

    PubMed

    He, Ran; Yang, Xinxin; Liu, Cheng; Chen, Xiangyu; Wang, Lin; Xiao, Minglu; Ye, Jianqiang; Wu, Yuzhang; Ye, Lilin

    2017-03-13

    CD4(+) T cells are essential for sustaining CD8(+) T cell responses during a chronic infection. The adoptive transfer of virus-specific CD4(+) T cells has been shown to efficiently rescue exhausted CD8(+) T cells. However, the question of whether endogenous virus-specific CD4(+) T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8(+) T cells remains unexplored. In this study, we developed a CD4(+) T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-A(b)-restricted CD4(+) T cell epitope GP61-80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector that expressed the same CD4(+) T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4(+) T cell responses, which further improved both the quantity and quality of the virus-specific CD8(+) T cells and led to better control of the viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced the anti-viral CD8(+) T cell responses and viral clearance. Thus, a heterologous prime-boost immunization that selectively induces virus-specific CD4(+) T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.Cellular & Molecular Immunology advance online publication, 13 March 2017; doi:10.1038/cmi.2017.3.

  14. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  15. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the Envelope Glycoprotein Gene Sequence.

    DTIC Science & Technology

    1991-11-29

    AD-A261 707 AD____ ARMY PROJECT ORDER NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE...DATES COVERED 29 Nov 91 Final Report (9/1/89 - 11/30/91) 4. TITLE AND SUBTITLE Ana ysis or Dengue Vnrus nancing 5. FUNDING NUMBERS Epitopes Using...biological events leading to the development of severe disease manifestations of dengue infections ( dengue hemorrhagic fever/ dengue shock syndrome

  16. Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived from the Envelope Glycoprotein Gene Sequence

    DTIC Science & Technology

    1990-11-27

    AD________ AD-A230 976 ARMY PROJECT NO: 89PP9961 TITLE: ANALYSIS OF DENGUE VIRUS ENHANCING EPITOPES USING PEPTIDE ANTIGENS DERIVED FROM THE ENVELOPE...INO. INO r CCESSION NO I1I TITLE (Include Security Classification) Analysis of Dengue Virus Enhancing Epitopes Using Peptide Antigens Derived From the...necessary and identify by block number) Antibody-dependent enhancement (ADE) ot dengue (DEN) virus infection in human mononuclear cells in vitro has been

  17. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance

    PubMed Central

    van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H. J.; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines. PMID:27333291

  18. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

    PubMed

    Rosendahl Huber, Sietske K; Luimstra, Jolien J; van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H J; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

  19. Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

    PubMed

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2015-04-01

    Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein.

  20. Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys.

    PubMed

    Herrera, S; De Plata, C; González, M; Perlaza, B L; Bettens, F; Corradin, G; Arévalo-Herrera, M

    1997-04-01

    Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS(p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitro gamma-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or gamma-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitro gamma-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.

  1. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  2. Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

    PubMed

    Berger, Peter; Lapthorn, Adrian J

    2016-01-01

    hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

  3. Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

    PubMed Central

    Pan, Keyao; Long, Jinxue; Sun, Haoxin; Tobin, Gregory J.; Nara, Peter L.

    2010-01-01

    The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R2 > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9405-4) contains supplementary material, which is available to authorized users. PMID:21086120

  4. Mapping Antigenic Epitopes on the Human Bocavirus Capsid

    PubMed Central

    Kailasan, Shweta; Garrison, Jamie; Ilyas, Maria; Chipman, Paul; McKenna, Robert; Kantola, Kalle; Söderlund-Venermo, Maria; Kučinskaitė-Kodzė, Indrė; Žvirblienė, Aurelija

    2016-01-01

    ABSTRACT Human bocaviruses (HBoV1 to -4) are emerging pathogens associated with pneumonia and/or diarrhea in young children. Currently, there is no treatment or vaccination, so there is a need to study these pathogens to understand their disease mechanisms on a molecular and structural level for the development of control strategies. Here, we report the structures of six HBoV monoclonal antibody (MAb) fragment complexes, HBoV1-15C6, HBoV2-15C6, HBoV4-15C6, HBoV1-4C2, HBoV1-9G12, and HBoV1-12C1, determined by cryo-electron microscopy and three-dimensional image reconstruction to 18.0- to 8.5-Å resolution. Of these, the 15C6 MAb cross-reacted with HBoV1, HBoV2, and HBoV4, while the 4C2, 12C1, and 9G12 MAbs recognized only HBoV1. Pseudoatomic modeling mapped the 15C6 footprint to the capsid surface DE and HI loops, at the 5-fold axis and the depression surrounding it, respectively, which are conserved motifs in Parvoviridae. The footprints for 4C2, 12C1, and 9G12 span the surface loops that assemble portions of the 2-/5-fold wall (a raised surface feature between the 2-fold and 5-fold axes of symmetry) and the shoulder of the 3-fold protrusions. The MAb footprints, cross reactive and strain specific, coincide with regions with high and low sequence/structural identities, respectively, on the capsid surfaces of the HBoVs and identify potential regions for the development of peptide vaccines for these viruses. IMPORTANCE Human bocaviruses (HBoVs) may cause severe respiratory and gastrointestinal infections in young children. The nonenveloped parvovirus capsid carries determinants of host and tissue tropism, pathogenicity, genome packaging, assembly, and antigenicity important for virus infection. This information is currently unavailable for the HBoVs and other bocaparvoviruses. This study identifies three strain-specific antigenic epitopes on the HBoV1 capsid and a cross-reactive epitope on the HBoV1, HBoV2, and HBoV4 capsids using structures of capsid

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

    DOE PAGES

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

    2016-05-18

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

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

    SciTech Connect

    Chen, Edwin; Salinas, Nichole D.; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D.; Gross, Michael L.; Adams, John H.; Tolia, Niraj Harish

    2016-05-18

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

  7. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    NASA Astrophysics Data System (ADS)

    Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

    1994-06-01

    IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

  8. Identification of Enteroviruses by Using Monoclonal Antibodies against a Putative Common Epitope

    PubMed Central

    Shin, Soo-Youn; Kim, Ki-Soon; Lee, Yoon-Sung; Chung, Yoon-Seok; Park, Kwi-Sung; Cheon, Doo-Sung; Na, Byoung-Kuk; Kang, Yoonsung; Cheong, Hyang-Min; Moon, Youngjoon; Choi, Jee-Hye; Cho, Hang-Eui; Min, Na-Young; Son, Jin-Sook; Park, Young-Hoon; Jee, Youngmee; Yoon, Jae-Deuk; Song, Chul-Yong; Lee, Kwang-Ho

    2003-01-01

    A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses. PMID:12843038

  9. Whole-exome sequencing predicted cancer epitope trees of 23 early cervical cancers in Chinese women.

    PubMed

    Li, Xia; Huang, Hailiang; Guan, Yanfang; Gong, Yuhua; He, Cheng-Yi; Yi, Xin; Qi, Ming; Chen, Zhi-Ying

    2017-01-01

    Emerging evidence suggest that the heterogeneity of cancer limits the efficacy of immunotherapy. To search for optimal therapeutic targets for enhancing the efficacy, we used whole-exome sequencing data of 23 early cervical tumors from Chinese women to investigate the hierarchical structure of the somatic mutations and the neo-epitopes. The putative neo-epitopes were predicted based on the mutant peptides' strong binding with major histocompatibility complex class I molecules. We found that each tumor carried an average of 117 mutations and 61 putative neo-epitopes. Each patient displayed a unique phylogenetic tree in which almost all subclones harbored neo-epitopes, highlighting the importance of individual neo-epitope tree in determination of immunotherapeutic targets. The alterations in FBXW7 and PIK3CA, or other members of the significantly altered ubiquitin-mediated proteolysis and extracellular matrix receptor interaction related pathways, were proposed as the earliest changes triggering the malignant progression. The neo-epitopes involved in these pathways, and located at the top of the hierarchy tree, might become the optimal candidates for therapeutic targets, possessing the potential to mediate T-cell killing of the descendant cells. These findings expanded our understanding in early stage of cervical carcinogenesis and offered an important approach to assist optimizing the immunotherapeutic target selection.

  10. Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers.

    PubMed Central

    Pennell, RI; Janniche, L; Kjellbom, P; Scofield, GN; Peart, JM; Roberts, K

    1991-01-01

    We have identified and characterized the temporal and spatial regulation of a plasma membrane arabinogalactan protein epitope during development of the aerial parts of oilseed rape using the monoclonal antibody JIM8. The JIM8 epitope is expressed by the first cells of the embryo and by certain cells in the sexual organs of flowers. During embryogenesis, the JIM8 epitope ceases to be expressed by the embryo proper but is still found in the suspensor. During differentiation of the stamens and carpels, expression of the JIM8 epitope progresses from one cell type to another, ultimately specifying the endothecium and sperm cells, the nucellar epidermis, synergid cells, and the egg cell. This complex temporal sequence demonstrates rapid turnover of the JIM8 epitope. There is no direct evidence for any cell-inductive process in plant development. However, if cell-cell interactions exist in plants and participate in flower development, the JIM8 epitope may be a marker for one set of them. PMID:12324592

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

    PubMed

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

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

  12. Identification and Phylogeny of the First T Cell Epitope Identified from a Human Gut Bacteroides Species

    PubMed Central

    Perez-Muñoz, Maria Elisa; Joglekar, Payal; Shen, Yi-Ji; Chang, Kuan Y.; Peterson, Daniel A.

    2015-01-01

    Host T cell reactivity toward gut bacterial epitopes has been recognized as part of disease pathogenesis. However, the specificity of T cells that recognize this vast number of epitopes has not yet been well described. After colonizing a C57BL/6J germ-free mouse with the human gut symbiotic bacteria Bacteroides thetaiotaomicron, we isolated a T cell that recognized these bacteria in vitro. Using this T cell, we mapped the first known non-carbohydrate T cell epitope within the phylum Bacteroidetes. The T cell also reacted to two other additional Bacteroides species. We identified the peptide that stimulated the T cell by using a genetic approach. Genomic data from the epitope-positive and epitope-negative bacteria explain the cross-reactivity of the T cell to multiple species. This epitope degeneracy should shape our understanding of the T cell repertoire stimulated by the complex microbiome residing in the gastrointestinal tract in both healthy and disease states. PMID:26637014

  13. Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid

    PubMed Central

    Parker, Tracy Dewese; Kitamoto, Noritoshi; Tanaka, Tomoyuki; Hutson, Anne M.; Estes, Mary K.

    2005-01-01

    Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay. PMID:15919896

  14. Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes

    PubMed Central

    Schaubert, Keri L.; Price, David A.; Salkowitz, Janelle R.; Sewell, Andrew K.; Sidney, John; Asher, Tedi E.; Blondelle, Sylvie E.; Adams, Sharon; Marincola, Francesco M.; Joseph, Aviva; Sette, Alessandro; Douek, Daniel C.; Ayyavoo, Velpandi; Storkus, Walter; Leung, Ming-Ying; Ng, Hwee L.; Yang, Otto O.; Goldstein, Harris; Wilson, Darcy B.; Kan-Mitchell, June

    2011-01-01

    Summary HLA-A*0201-restricted virus-specific CD8+ cytotoxic T lymphocytes (CTLs) do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag19–27), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9-specific CTLs primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA-A*0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro-primed CTLs showed that TV9p6 consistently activated crossreactive and equally functional CTLs as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTLs primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a “public” TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional crossreactive clonotypes that are less readily recruited from the naïve T cell pool by the corresponding wildtype epitope. Mimotope-induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine-induced responses to conserved but poorly immunogenic determinants within the HIV proteome. PMID:20432235

  15. Cloning of the first invertebrate MAGE paralogue: an epitope that activates T-cells in humans is highly conserved in evolution.

    PubMed

    Põld, M; Põld, A; Ma, H J; Sjak-Shieb, N N; Vescio, R A; Berensonb, J R

    2000-12-01

    The MAGE (Melanoma Associated Antigen) family tumor-specific antigens are shared by a number of histologically different tumors. Till date, only human and mouse MAGE genes have been characterized. Our study describes the first non-mammalian member of MAGE super-family, DMAGE from D. melanogaster. A conceptual translation of the cDNA of DMAGE identifies a putative protein that contains a motif that shares eight out of nine amino acids with the previously identified promiscuous, HLA-A2 restricted antigenic epitope in the C-terminus of human MAGE-B1 and -B2. Similarly, this motif of DMAGE shares seven out of nine amino acids with the same antigenic epitope of human MAGE-A3 and -A12. Thus, the phylogeny of proteins that activate tumor specific T-cells in mammals as unmutated self-proteins began at least 100 million years earlier in evolution than the emergence of the adaptive immune system of higher vertebrates. Northern analysis revealed that DMAGE is a developmentally regulated gene highly expressed in adult fruit fly and in the embryo of D. melanogaster. In contrast, the expression level of the mRNA of DMAGE in fruit fly larva is substantially lower than in embryo and adult fly. We propose that studies of DMAGE on D. melanogaster may help define the function(s) of MAGE super-family genes.

  16. Exposure to Melan-A/MART-126-35 tumor epitope specific CD8(+)T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS).

    PubMed

    Ebstein, Frédéric; Keller, Martin; Paschen, Annette; Walden, Peter; Seeger, Michael; Bürger, Elke; Krüger, Elke; Schadendorf, Dirk; Kloetzel, Peter-M; Seifert, Ulrike

    2016-05-04

    Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

  17. Exposure to Melan-A/MART-126-35 tumor epitope specific CD8+T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS)

    PubMed Central

    Ebstein, Frédéric; Keller, Martin; Paschen, Annette; Walden, Peter; Seeger, Michael; Bürger, Elke; Krüger, Elke; Schadendorf, Dirk; Kloetzel, Peter-M.; Seifert, Ulrike

    2016-01-01

    Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing. PMID:27143649

  18. Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4.

    PubMed

    Kurane, I; Zeng, L; Brinton, M A; Ennis, F A

    1998-01-20

    The role of dengue virus-specific serotype-cross-reactive T lymphocytes in recovery from and pathogenesis of dengue virus infections is not known. In the present paper, we have defined a dengue serotype-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK36 and JK46. These T cell clones were established from the peripheral blood T lymphocytes of a dengue-3-immune donor, using a limiting dilution method. JK36 and JK46 were cross-reactive for dengue virus types 2, 3, and 4, but not for type 1, and recognized the NS3 protein. The smallest synthetic peptide recognized by JK36 was an 8-amino acid peptide that contains amino acids (aa) 226 to 233 (VVAAEMEE) of NS3. The smallest peptide recognized by JK46 was an 11-amino acid peptide that contains aa 224 to 234 (TRVVAAEMEEA). HLA-DR15 was the restriction allele for recognition of these peptides by both JK36 and JK46. This is the first epitope to be defined that is recognized by human CD4+ CTL cross-reactive for dengue virus types 2, 3, and 4.

  19. Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis.

    PubMed Central

    Hochleitner, E. O.; Borchers, C.; Parker, C.; Bienstock, R. J.; Tomer, K. B.

    2000-01-01

    A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope. PMID:10752610

  20. Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis.

    PubMed

    Hochleitner, E O; Borchers, C; Parker, C; Bienstock, R J; Tomer, K B

    2000-03-01

    A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.

  1. Epitope mapping of Canine distemper virus phosphoprotein by monoclonal antibodies.

    PubMed

    Sugai, Akihiro; Kooriyama, Takanori; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

    2009-12-01

    The gene for phosphoprotein (P) of CDV encodes three different proteins, P, V, and C. The P protein is involved in viral gene transcription and replication. In the present study, we produced MAbs against a unique domain of the CDV-P protein, from aa 232 to 507, and determined their antigenic sites. By immunizing BALB/c mice with the recombinant P protein-specific fragment, we obtained six MAbs. Competitive binding inhibition assays revealed that they recognized two distinct regions of the P protein. Western blot analysis and immunofluorescence assays using deletion mutants of the unique C-terminus of the CDV-P protein revealed that all MAbs recognized a central short region (aa 233-303) of the CDV-P protein. In addition, linear and conformational epitopes have been determined, and at least four antigenic sites exist in the P protein central region. Furthermore, four of the MAbs were found to react with the P protein of recent Japanese field isolates but not with that of the older CDV strains, including a vaccine strain. Thus, these MAbs could be clinically useful for quick diagnosis during the CDV outbreaks.

  2. Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus

    PubMed Central

    Chaudhury, Sidhartha; Gromowski, Gregory D.; Ripoll, Daniel R.; Khavrutskii, Ilja V.; Desai, Valmik; Wallqvist, Anders

    2017-01-01

    Background A majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response. Methods/Principal findings We hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III. Conclusions/Significance Our results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response

  3. Genetics Home Reference: familial restrictive cardiomyopathy

    MedlinePlus

    ... Home Health Conditions familial restrictive cardiomyopathy familial restrictive cardiomyopathy Enable Javascript to view the expand/collapse boxes. ... PDF Open All Close All Description Familial restrictive cardiomyopathy is a genetic form of heart disease. For ...

  4. An immunodominant HLA-A*1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients.

    PubMed

    Chen, Xiaoling; Wang, Wenbo; Wang, Shufeng; Meng, Gang; Zhang, Mengjun; Ni, Bing; Wu, Yuzhang; Wang, Li

    2013-12-01

    Hepatitis B virus (HBV) infection is a worldwide public health problem. HBV-specific CD8(+) CTLs are vital for viral clearance. Identification of immunodominant CTL epitopes from HBV-associated antigens is necessary for therapeutic vaccine development. We showed that the HLA-A*1101 allele is one of the most common alleles in both healthy individuals and chronic hepatitis B (CHB) patients in the Chongqing area, China. However, less than 10% of epitopes of HBV-associated antigens have been identified in an HLA-A*1101 context. Here, we describe an immunodominant CD8(+) T-cell response targeting a hepatitis B surface antigen determinant (HBs(295-304)) restricted by HLA-A*1101 in both healthy individuals and CHB patients. Moreover, HBs(295-304) is more immunogenic for CTL induction than a known naturally HLA-A*1101-processed epitope from hepatitis B core antigen (HBc(88-96)). Therefore, the newly identified epitope, HBs(295-304), will benefit the development of immunotherapeutic approaches for HBV infection.

  5. Harnessing Computational Biology for Exact Linear B-Cell Epitope Prediction: A Novel Amino Acid Composition-Based Feature Descriptor.

    PubMed

    Saravanan, Vijayakumar; Gautham, Namasivayam

    2015-10-01

    Proteins embody epitopes that serve as their antigenic determinants. Epitopes occupy a central place in integrative biology, not to mention as targets for novel vaccine, pharmaceutical, and systems diagnostics development. The presence of T-cell and B-cell epitopes has been extensively studied due to their potential in synthetic vaccine design. However, reliable prediction of linear B-cell epitope remains a formidable challenge. Earlier studies have reported discrepancy in amino acid composition between the epitopes and non-epitopes. Hence, this study proposed and developed a novel amino acid composition-based feature descriptor, Dipeptide Deviation from Expected Mean (DDE), to distinguish the linear B-cell epitopes from non-epitopes effectively. In this study, for the first time, only exact linear B-cell epitopes and non-epitopes have been utilized for developing the prediction method, unlike the use of epitope-containing regions in earlier reports. To evaluate the performance of the DDE feature vector, models have been developed with two widely used machine-learning techniques Support Vector Machine and AdaBoost-Random Forest. Five-fold cross-validation performance of the proposed method with error-free dataset and dataset from other studies achieved an overall accuracy between nearly 61% and 73%, with balance between sensitivity and specificity metrics. Performance of the DDE feature vector was better (with accuracy difference of about 2% to 12%), in comparison to other amino acid-derived features on different datasets. This study reflects the efficiency of the DDE feature vector in enhancing the linear B-cell epitope prediction performance, compared to other feature representations. The proposed method is made as a stand-alone tool available freely for researchers, particularly for those interested in vaccine design and novel molecular target development for systems therapeutics and diagnostics: https://github.com/brsaran/LBEEP.

  6. Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

    PubMed Central

    Sedegah, Martha; Hollingdale, Michael R.; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Kim, Yohan; Peters, Bjoern; Sette, Alessandro; Huang, Jun; McGrath, Shannon; Abot, Esteban; Limbach, Keith; Shi, Meng; Soisson, Lorraine; Diggs, Carter; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E.; Villasante, Eileen; Richie, Thomas L.

    2014-01-01

    Background Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. Methodology/Principal Findings We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. Conclusions/Significance We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches

  7. Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

    PubMed Central

    Shirai, M; Akatsuka, T; Pendleton, C D; Houghten, R; Wychowski, C; Mihalik, K; Feinstone, S; Berzofsky, J A

    1992-01-01

    Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the

  8. The Role of Glutamic or Aspartic Acid in Position Four of the Epitope Binding Motif and Thyrotropin Receptor-Extracellular Domain Epitope Selection in Graves' Disease

    PubMed Central

    Inaba, Hidefumi; Martin, William; Ardito, Matt; De Groot, Anne Searls; De Groot, Leslie J.

    2010-01-01

    Context: Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. Objective and Design: We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. Results: Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E−) (IC50 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E− peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E− peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. Conclusions: TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E− and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells. PMID:20392871

  9. 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.

  10. Limited conformational flexibility in the paratope may be responsible for degenerate specificity of HIV epitope recognition.

    PubMed

    Bhowmick, Arijit; Salunke, Dinakar M

    2013-02-01

    Exquisite specificity is the hallmark of antigen-antibody recognition. However, breakdown in the specific recognition potential culminating in the binding to multiple antigens by a single antibody has been observed, even after the maturation of the humoral response. While such a broad specificity may be expected to assist the host to counter the antigenic variations associated with an immune-evading pathogen, escape from immune surveillance by subtle epitopic mutations in pathogens like HIV and influenza virus has been clearly established. In the light of this dichotomy, the issues of degeneracy/specificity in the humoral response against such epitopes were analysed using three HIV-neutralizing epitopes and their variants as a model system. Cross-reactivity was observed in the polyclonal response against two of the epitopes. Multi-reactive mAb KEL10 was isolated against one of the epitopes, ELDKWA from this response. It is evident that even after the affinity maturation, antibodies showing binding to multiple variants of an immunizing peptide epitope existed. Binding kinetics and in silico structural analyses indicated that conserved interactions across epitopes and limited conformational flexibility in the paratope may account for the observed multi-reactivity. Though the affinity maturation process is expected to incorporate an extent of specificity to the paratope, there appear to be still some B-cell clones producing antibodies with subtle flexibility in their binding site, as demonstrated in case of KEL10. Generation of such antibodies against effective immunogens could be a possible approach for countering the antibody neutralization escape by various immune-evading pathogens.

  11. Mapping of SLE-specific Sm B cell epitopes using murine monoclonal antibodies.

    PubMed

    Pruijn, G J; Schoute, F; Thijssen, J P; Smeenk, R J; van Venrooij, W J

    1997-04-01

    In this study we have used a number of monoclonal antibodies with various anti-Sm specificities originating from MRL/lpr mice to map B cell epitopes of the Sm-B/B' and Sm-D1 proteins. Selection of Sm-B subfragments reactive with the Sm-B/B'-specific monoclonal antibody ANA125 from a DNaseI fragment expression library revealed that the epitope recognized by this monoclonal antibody is located between amino acids 146 and 158: GRGTVAAAAAAAT. The epitopes recognized by two distinct Sm-D1-specific monoclonal antibodies, 7.13 and ANA127, appeared to be located in the carboxy-terminal region of the protein as revealed by immunoprecipitation of in vitro translated deletion mutants of Sm-D1. These epitopes are probably identical and not simply composed of a GR repeat, which is a characteristic feature of this part of the protein. Immunoprecipitation of in vitro translated deletion mutants of both Sm-B and Sm-D1 was also employed to determine the sequence requirements for recognition by two monoclonal antibodies that are cross-reactive with several Sm proteins, Y12 and ANA128. The epitope recognized by these two monoclonal antibodies is probably also identical and composed by the juxtaposition of several regions in the folded protein. The low, but significant, level of immunoprecipitation of truncated versions of both Sm-B and Sm-D1, suggests that the Sm domain, which is shared by all Sm proteins, in particular the amino-terminal part of the Sm1 motif of Sm-B and Sm-D1, plays an important role in formation of the cross-reactive epitope and might contribute to cross-reactivity with other Sm proteins. The results of immunoprecipitation experiments with cellular extracts show that the epitopes recognized by all anti-Sm monoclonal antibodies used in this study are accessible in the assembled snRNPs.

  12. Towards the knowledge-based design of universal influenza epitope ensemble vaccines

    PubMed Central

    Sheikh, Qamar M.; Gatherer, Derek; Reche, Pedro A; Flower, Darren R.

    2016-01-01

    Motivation: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. Results: To exemplify our approach we designed two epitope ensemble vaccines comprising highly conserved and experimentally verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96 and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97 and 88% coverage of observed subtypes. Availability and Implementation: http://imed.med.ucm.es/Tools/episopt.html. Contact: d.r.flower@aston.ac.uk PMID:27402904

  13. A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice.

    PubMed

    Tu, Jianxin; Hou, Bailong; Wang, Bingbing; Lin, Xiaoyun; Gong, Wenci; Dong, Haiyan; Zhu, Shanli; Chen, Shao; Xue, Xiangyang; Zhao, Kong-Nan; Zhang, Lifang

    2014-05-01

    We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) multi-epitope of Chlamydia trachomatis. A short gene of multi-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a(+) containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His-MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multi-epitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies (IgG1 and IgG2a), but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi-epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Th1 response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi-epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.

  14. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  15. Maternal food restrictions during breastfeeding

    PubMed Central

    Jeong, Goun; Park, Sung Won; Lee, Yeon Kyung; Ko, Sun Young

    2017-01-01

    Purpose This study investigated self-food restriction during breastfeeding, reviewed the literature showing the effect of maternal diet on the health of breast-fed infants, and explored the validity of dietary restrictions. Methods Questionnaire data were collected from breastfeeding Korean mothers who visited the pediatric clinic of Cheil General Hospital & Women's Healthcare Center from July 2015 through August 2015. The survey included items assessing maternal age, number of children, maternal educational attainment, household income, degree of difficulty with self-food restriction, types of self-restricted foods, dietary customs during breastfeeding, and sources of information about breastfeeding. Results The questionnaire was completed by 145 mothers. More than a third (n=56, 39%) had discomfort from and usually avoided 4–5 types of food (mean, 4.92). Mothers younger than 40 years had more discomfort (odds ratio [OR], 12.762; P=0.017). Primiparas felt less discomfort than multiparas (OR, 0.436; P=0.036). Dietary practices were not influenced by maternal educational attainment or household income. The most common self-restricted foods were caffeine (n=131, 90.3%), spicy foods (n=124, 85.5%), raw foods (n=109, 75.2%), cold foods (n=100, 69%), and sikhye (traditional sweet Korean rice beverage) (n=100, 69%). Most mothers (n=122, 84.1%) avoided foods for vague reasons. Conclusion Most mothers restricted certain foods unnecessarily. Literature review identified no foods that mothers should absolutely avoid during breastfeeding unless the infant reacts negatively to the food. PMID:28392822

  16. Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice

    PubMed Central

    Escalona, Emilia; Sáez, Darwin; Oñate, Angel

    2017-01-01

    Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus. PMID:28232837

  17. The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors.

    PubMed

    Kawashima, I; Hudson, S J; Tsai, V; Southwood, S; Takesako, K; Appella, E; Sette, A; Celis, E

    1998-01-01

    One approach to development of specific cancer immunotherapy relies on the induction of cytotoxic T lymphocytes (CTL) specific for tumor-associated antigens (TAA). Induction of TAA-specific CTL could be used towards the eradication of established tumors, or to prevent their dissemination or recurrence after primary treatment. The present study identifies a set of CTL epitopes from TAA frequently found on solid epithelial tumors such as breast, lung and gastro-intestinal tumors. Specifically, HLA-A2.1 binding peptides from the MAGE2, MAGE3, HER-2/neu and CEA antigens were tested for their capacity to elicit in vitro anti-tumor CTL using lymphocytes from normal volunteers and autologous dendritic cells as antigen-presenting cells. A total of 6 new epitopes (MAGE2[10(157)], MAGE3[9(112)], CEA[9(691)], CEA[9(24)], HER2[9(435)] and HER2[9(5)]) were identified which were capable of specifically recognizing tumor cell lines lines expressing HLA-A2.1 and the corresponding TAA. In one case (CEA[9(24)]), induction of vigorous anti-tumor CTL responses required epitope engineering to increase HLA-A2.1 binding affinity. Finally, most of the newly identified epitopes (5 out of 6) were found to be highly crossreactive with other common HLA alleles of the A2 supertype (A2.2, A2.3, A2.6 and A6802), thus demonstrating their potential in providing broad and non-ethnically biased population coverage. The results are discussed in the context of the development of multi-epitope-based therapies with broad applicability for patients suffering from commonly found tumors.

  18. Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu-Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice.

    PubMed

    Escalona, Emilia; Sáez, Darwin; Oñate, Angel

    2017-01-01

    Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu-Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4(+) T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus.

  19. Isolation of human CD4/CD8 double-positive, graft-versus-host disease-protective, minor histocompatibility antigen-specific regulatory T cells and of a novel HLA-DR7-restricted HY-specific CD4 clone.

    PubMed

    Eljaafari, Assia; Yuruker, Ozel; Ferrand, Christophe; Farre, Annie; Addey, Caroline; Tartelin, Marie-Laure; Thomas, Xavier; Tiberghien, Pierre; Simpson, Elizabeth; Rigal, Dominique; Scott, Diane

    2013-01-01

    Minor histocompatibility (H) Ags are classically described as self-peptides derived from intracellular proteins that are expressed at the cell surface by MHC class I and class II molecules and that induce T cell alloresponses. We have isolated three different T cell populations from a skin biopsy of a patient suffering from acute graft-versus-host disease following sex-mismatched HLA-identical bone marrow transplantation. The first population was: 1) CD4(+)/CD8(+) double-positive; 2) specific for an HLA class I-restricted autosomal Ag; 3) expressed a Tr1 profile with high levels of IL-10, but low IL-2 and IFN-γ; and 4) exerted regulatory function in the presence of recipient APCs. The second was CD8 positive, specific for an HLA class I-restricted autosomally encoded minor H Ag, but was only weakly cytotoxic. The third was CD4 single positive, specific for an HLA-DR7-restricted HY epitope and exerted both proliferative and cytotoxic functions. Identification of the peptide recognized by these latter cells revealed a new human HY epitope, TGKIINFIKFDTGNL, encoded by RPS4Y and restricted by HLA-DR7. In this paper, we show human CD4/CD8 double-positive, acute graft-versus-host disease-protective, minor H Ag-specific regulatory T cells and identify a novel HLA-DR7/ HY T cell epitope, encoded by RPS4Y, a potential new therapeutic target.

  20. Restricted autoantigen recognition associated with deletional and adaptive regulatory mechanisms.

    PubMed

    Gebe, John A; Yue, Betty B; Unrath, Kelly A; Falk, Ben A; Nepom, Gerald T

    2009-07-01

    Autoimmune diabetes (T1D) is characterized by CD4(+) T cell reactivity to a variety of islet-associated Ags. At-risk individuals, genetically predisposed to T1D, often have similar T cell reactivity, but nevertheless fail to progress to clinically overt disease. To study the immune tolerance and regulatory environment permissive for such autoreactive T cells, we expressed TCR transgenes derived from two autoreactive human T cells, 4.13 and 164, in HLA-DR4 transgenic mice on a C57BL/6-derived "diabetes-resistant" background. Both TCR are responsive to an immunodominant epitope of glutamic acid decarboxylase 65(555-567), which is identical in sequence between humans and mice, is restricted by HLA-DR4, and is a naturally processed self Ag associated with T1D. Although both TCR use the identical Valpha and Vbeta genes, differing only in CDR3, we found stark differences in the mechanisms utilized in vivo in the maintenance of immune tolerance. A combination of thymic deletion (negative selection), TCR down-regulation, and peripheral activation-induced cell death dominated the phenotype of 164 T cells, which nevertheless still maintain their Ag responsiveness in the periphery. In contrast, 4.13 T cells are much less influenced by central and deletional tolerance mechanisms, and instead display a peripheral immune deviation including differentiation into IL-10-secreting Tr1 cells. These findings indicate a distinct set of regulatory alternatives for autoreactive T cells, even within a single highly restricted HLA-peptide-TCR recognition profile.

  1. Neutralizing activity of antibodies to the V3 loop region of HIV-1 gp120 relative to their epitope fine specificity

    PubMed Central

    Pantophlet, Ralph; Wrin, Terri; CavaciniJ, Lisa A.; Robinson, James E.; Burton, Dennis R.

    2008-01-01

    The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2–3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR β hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information suggests that an antibody’s mode of interaction with V3, driven by contact residue requirements, precludes the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses. PMID:18822440

  2. Functional profile of human influenza virus-specific cytotoxic T lymphocyte activity is influenced by interleukin-2 concentration and epitope specificity

    PubMed Central

    Boon, ACM; de Mutsert, G; Fouchier, RAM; Osterhaus, ADME; Rimmelzwaan, GF

    2005-01-01

    The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8+ T cell populations specific for the HLA-A*0101 restricted epitope NP44–52 and the HLA-A*0201 restricted epitope M158–66 were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-γ and tumour necrosis factor-(TNF)-α upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-γ and TNF-α by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm+ and M1-Tm+ cells differed considerably. Overall, no difference in the proportion of NP-Tm+ or M1-Tm+ cells expressing CD107a was observed. The proportion of M1-Tm+ cells that produced IFN-γ (P < 0·05) was larger than for NP-Tm+ cells, independent of IL-2 concentration. When cultured under IL-2hi concentrations higher TNF-α expression was also observed in M1-Tm+ cells (P < 0·05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm+ and NP-Tm+ cells. PMID:16178855

  3. Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection.

    PubMed

    Song, Xiaokai; Xu, Lixin; Yan, Ruofeng; Huang, Xinmei; Li, Xiangrui

    2015-08-15

    The search for effective vaccines against chicken coccidiosis remains a challenge because of the complex organisms with multiple life cycle stages of Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle could be an optimal strategy to overcome the antigen complexity of the parasite. In this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic expression vector pVAX1 in different forms, with or without chicken cytokines IL-2 or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA vaccines against Eimeria tenella. Transcription and expression of the multi-epitope DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried out to evaluate the protective efficacy against Eimeria tenella. Results showed the constructed DNA vaccines were transcribed and translated successfully in vivo. Animal experiment showed that the multi-epitopes DNA vaccines were more effective to stimulate immune response than single fragment. Compared with the DNA vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2 containing 4 fragments with concentrated T-epitopes provided the highest ACI of 180.39. DNA vaccines composed of antigens from two developmental stages were more effective than the single-stage ones. Especially DNA vaccine pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39. Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most effective protection with the ACI of 189.92. The multi-epitope DNA vaccines revealed in this study provide new candidates for Eimeria vaccine development.

  4. Receptor epitope usage by an interleukin-5 mimetic peptide.

    PubMed

    Ishino, Tetsuya; Urbina, Cecilia; Bhattacharya, Madhushree; Panarello, Dominick; Chaiken, Irwin

    2005-06-17

    The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.

  5. Generalized Pump-restriction Theorem

    SciTech Connect

    Sinitsyn, Nikolai A; Chernyak, Vladimir Y

    2008-01-01

    We formulate conditions under which periodic modulations of parameters on a finite graph with stochastic transitions among its nodes do not lead to overall pump currents through any given link. Our theorem unifies previously known results with the new ones and provides a universal approach to explore futher restrictions on stochastic pump effect in non-adiabatically driven systems with detailed balance.

  6. Cloning, molecular analysis and epitopics prediction of a new chaperone GroEL Brucella melitensis antigen

    PubMed Central

    Sekhavati, Mohammad Hadi; Heravi, Reza Majidzadeh; Tahmoorespur, Mojtaba; Yousefi, Soheil; Abbassi-Daloii, Tooba; Akbari, Rahebe

    2015-01-01

    Objective(s): Brucellosis is a well-known domestic animal infectious disease, which is caused by Brucella bacterium. GroEL antigen increases Brucella survival and is one of the major antigens that stimulates the immune system. Hence, the objective of the present study was cloning and bioinformatics analysis of GroEL gene. Materials and Methods: The full-length open reading frame of this gene was amplified by specific primers and cloned into pTZ57R/T vector. Also, the sequence of this gene in the Brucella melitensis strain Rev 1 was submitted to the NCBI gene bank for the first time. Several prediction software applications were also used to predict B and T-cell epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. The used software applications validated experimental results. Results: The results of phylogenetic analysis showed that the GroEL sequence had near homology with other species instead of other Brucella spp. The bioinformatics tools used in the current study were validated by the results of four different experimental epitope predictions. Bioinformatics analysis identified eight B and seven T-cell epitopes. Conclusion: According to the antigenic ability and proteasomal cleavage sites, four (150-160, 270-285,351-361 and 385-395) common epitopes were predicted for GroEL gene. Bioinformatics analysis showed that these regions had proper epitope characterization and so may be useful for stimulation of cell-mediated and humoral immunity system. PMID:26124937

  7. PrP antibody binding-induced epitope modulation evokes immunocooperativity.

    PubMed

    Chang, Binggong; Miller, Michael W; Bulgin, Marie S; Sorenson-Melson, Sharon; Balachandran, Aru; Chiu, Allen; Rubenstein, Richard

    2008-12-15

    We have characterized the antibody-antigen binding events of the prion protein (PrP) utilizing three new PrP-specific monoclonal antibodies (Mabs). The degree of immunoreactivity was dependent on the denaturation treatment with the combination of heat and SDS resulting in the highest levels of epitope accessibility and antibody binding. Interestingly however, this harsh denaturation treatment was not sufficient to completely and irreversibly abolish protein conformation. The Mabs differed in their PrP epitopes with Mab 08-1/11F12 binding in the region of PrP(93-122), Mab 08-1/8E9 reacting to PrP(155-200) and Mab 08-1/5D6 directed to an undefined conformational epitope. Using normal and infected brains from hamsters, sheep and deer, we demonstrate that the binding of PrP to one Mab triggers PrP epitope unmasking, which enhances the binding of a second Mab. This phenomenon, termed positive immunocooperativity, is specific regarding epitope and the sequence of binding events. Positive immunocooperativity will likely increase immunoassay sensitivity since assay conditions for PrP(Sc) detection does not require protease digestion.

  8. Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.

    PubMed

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3(1073) and NS3(1446), and multiple variants for epitope NS3(1406) that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross-reactivity of vaccine-induced T cells was determined using variant peptides in IFN-γ ELISPOT assays. Vaccine-induced T cells targeted approximately 90% of NS3(1073) genotype 1 sequences and 50% of NS3(1446) genotype 1 and 3 sequences. For NS3(1406), 62% of subtype-1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3(1406) that was maximally cross-reactive. In vitro priming assays showed that of those tested the NS3(1406) vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross-reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross-reactive T cells at immunodominant epitopes. The degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.

  9. Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response.

    PubMed Central

    McLachlan, S M; Rapoport, B

    1995-01-01

    TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production. PMID:7544244

  10. Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes.

    PubMed

    Ballew, John T; Murray, Joseph A; Collin, Pekka; Mäki, Markku; Kagnoff, Martin F; Kaukinen, Katri; Daugherty, Patrick S

    2013-11-26

    To enable discovery of serum antibodies indicative of disease and simultaneously develop reagents suitable for diagnosis, in vitro directed evolution was applied to identify consensus peptides recognized by patients' serum antibodies. Bacterial cell-displayed peptide libraries were quantitatively screened for binders to serum antibodies from patients with celiac disease (CD), using cell-sorting instrumentation to identify two distinct consensus epitope families specific to CD patients (PEQ and (E)/DxFV(Y)/FQ). Evolution of the (E)/DxFV(Y)/FQ consensus epitope identified a celiac-specific epitope, distinct from the two CD hallmark antigens tissue transglutaminase-2 and deamidated gliadin, exhibiting 71% sensitivity and 99% specificity (n = 231). Expansion of the first-generation PEQ consensus epitope via in vitro evolution yielded octapeptides QPEQAFPE and PFPEQxFP that identified ω- and γ-gliadins, and their deamidated forms, as immunodominant B-cell epitopes in wheat and related cereal proteins. The evolved octapeptides, but not first-generation peptides, discriminated one-way blinded CD and non-CD sera (n = 78) with exceptional accuracy, yielding 100% sensitivity and 98% specificity. Because this method, termed antibody diagnostics via evolution of peptides, does not require prior knowledge of pathobiology, it may be broadly useful for de novo discovery of antibody biomarkers and reagents for their detection.

  11. Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1).

    PubMed

    Shigeta, M; Hasegawa, A; Hamada, Y; Koyama, K

    2000-07-01

    The zona pellucida (ZP) of mammalian oocytes forms an extracellular matrix composed of three major glycoproteins and plays an important role in sperm-zona interactions. As ZP had a strong organ-specific but species-cross-reactive antigenicity and passive or active immunization with ZP antigens could impair fertilization, the possibility of developing a immunocontraceptive vaccine has been extensively studied. Studies on active immunization with porcine ZP (pZP) that contain B cell epitopes and T cell epitopes demonstrated that a temporary infertility could be induced along with the elevation of antibody titers, but it was always associated with ovarian failure. This could be due to the oophoritis by activation of pathogenic T cell immunity. It is the general consideration that any adverse effects by vaccination should be avoided for an immunocontraception. From this point of view, the analysis of B cell epitopes of pZP protein would be helpful for construction of a safe immunocontraceptive vaccine with zona antigens. We determined the amino acid sequence of the B epitope in the pZP1 protein by using a monoclonal antibody (MAb-5H4) that possesses a fertilization blocking ability. In addition, antiserum raised to the epitope sequence was revealed to block in vitro fertilization of homologous animal species.

  12. Cross‐reactivity of hepatitis C virus specific vaccine‐induced T cells at immunodominant epitopes

    PubMed Central

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul

    2014-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS31073 and NS31446, and multiple variants for epitope NS31406 that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross‐reactivity of vaccine‐induced T cells was determined using variant peptides in IFN‐γ ELISPOT assays. Vaccine‐induced T cells targeted approximately 90% of NS31073 genotype 1 sequences and 50% of NS31446 genotype 1 and 3 sequences. For NS31406, 62% of subtype‐1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS31406 that was maximally cross‐reactive. In vitro priming assays showed that of those tested the NS31406 vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross‐reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross‐reactive T cells at immunodominant epitopes. The degree of cross‐reactivity varies between epitopes and may be HCV‐subtype specific. PMID:25263407

  13. Hepitopes: A live interactive database of HLA class I epitopes in hepatitis B virus

    PubMed Central

    2016-01-01

    Increased clinical and scientific scrutiny is being applied to hepatitis B virus (HBV), with focus on the development of new therapeutic approaches, ultimately aiming for cure. Defining the optimum natural CD8+ T cell immune responses that arise in HBV, mediated by HLA class I epitope presentation, may help to inform novel immunotherapeutic strategies. Therefore, we have set out to develop a comprehensive database of these epitopes in HBV, coined ‘Hepitopes’. This undertaking has its foundations in a systematic literature review to identify the sites and sequences of all published class I epitopes in HBV. We also collected information regarding the methods used to define each epitope, and any reported associations between an immune response to this epitope and disease outcome. The results of this search have been collated into a new open-access interactive database that is available at http://www.expmedndm.ox.ac.uk/hepitopes. Over time, we will continue to refine and update this resource, as well as inviting contributions from others in the field to support its development. This unique new database is an important foundation for ongoing investigations into the nature and impact of the CD8+ T cell response to HBV. PMID:27976751

  14. A promising multiple-epitope recombinant vaccine against classical swine fever virus.

    PubMed

    Tian, Hong; Hou, Xiangming; Wu, Jinyan; Chen, Yan; Shang, Youjun; Yin, Shuanghui; Zhang, Keshan; Liu, Xiangtao

    2014-01-15

    Classical swine fever (CSF) is a highly contagious and often fatal disease of swine. It is caused by classical swine fever virus (CSFV), one of the members of the genus Pestivirus of the Flaviviridae family. The development of a safe and effective vaccine against the CSF is critical to pandemic control, this article shows a tandem-repeat multiple-epitope recombinant vaccine can protect pigs from CSFV challenge. That was composed as following: two copies each of glycoprotein E2 residues 693-707, 241-276 and 770-781, and two copies amino acid residues 1446-1460 of the non-structural protein NS2-3. In the challenge test, all of the swine vaccinated with Chinese vaccine strain (C-strain) were fully protected from a challenge with CSFV. However, after three successive vaccinations with the multiple-epitope recombinant vaccine, three out of five pigs were protected from challenge with CSFV (in terms of both clinical signs and viremia). These results demonstrate that multiple-epitope recombinant vaccine which carrying the major CSFV epitopes can induce a high level of epitope-specific antibodies and exhibit a protective capability that parallels induced by C-strain to a certain extent.

  15. A novel monoclonal antibody to a defined peptide epitope in MUC16.

    PubMed

    Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa; Blixt, Ola; Arigi, Emma; Pereira, Daniela; Høgdall, Estrid; Mandel, Ulla; Bennett, Eric P; Vakhrushev, Sergey Y; David, Leonor; Clausen, Henrik

    2015-11-01

    The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.

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

    PubMed Central

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

    2014-01-01

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

  17. Sequence- and Structure-Based Immunoreactive Epitope Discovery for Burkholderia pseudomallei Flagellin

    PubMed Central

    Nithichanon, Arnone; Rinchai, Darawan; Gori, Alessandro; Lassaux, Patricia; Peri, Claudio; Conchillio-Solé, Oscar; Ferrer-Navarro, Mario; Gourlay, Louise J.; Nardini, Marco; Vila, Jordi; Daura, Xavier; Colombo, Giorgio; Bolognesi, Martino; Lertmemonkolchai, Ganjana

    2015-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T