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Sample records for mhc-i-restricted epitopes conserved

  1. 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.

  2. 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

  3. MHC-I restricted Melanoma Antigen Specific TCR Engineered Human CD4+ T Cells Exhibit Multifunctional Effector and Helper Responses, In Vitro

    PubMed Central

    Ray, Swagatam; Chhabra, Arvind; Chakraborty, Nitya G.; Hegde, Upendra; Dorsky, David I.; Chodon, Thinle; von Euw, Erika; Comin-Anduix, Begonya; Koya, Richard C.; Ribas, Antoni; Economou, James S.; Rosenberg, Steven A.; Mukherji, Bijay

    2010-01-01

    MHC class 1-restricted human melanoma epitope MART-127–35 specific TCR engineered CD4+CD25− T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25− T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-γ, TNF-α, IL-2, MIP1ß), lyse target cells, and “help” the expansion of the MART-127–35 specific CD8+ T cells when stimulated by the MART-127–35 peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-127–35 specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR-transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies. PMID:20547105

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus nucleoprotein.

    PubMed

    Yu, Ruisong; Fan, Xiaoming; Xu, Wanxiang; Li, Wentao; Dong, Shijuan; Zhu, Yumin; He, Yaping; Tang, Haiping; Du, Rong; Li, Zhen

    2015-01-30

    Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.

  15. 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

  16. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi.

    PubMed Central

    Rowland-Jones, S L; Dong, T; Fowke, K R; Kimani, J; Krausa, P; Newell, H; Blanchard, T; Ariyoshi, K; Oyugi, J; Ngugi, E; Bwayo, J; MacDonald, K S; McMichael, A J; Plummer, F A

    1998-01-01

    Many people who remain persistently seronegative despite frequent HIV exposure have HIV-specific immune responses. The study of these may provide information about mechanisms of natural protective immunity to HIV-1. We describe the specificity of cytotoxic T lymphocyte responses to HIV in seronegative prostitutes in Nairobi who are apparently resistant to HIV infection. These women have had frequent exposure to a range of African HIV-1 variants, primarily clades A, C, and D, for up to 12 yr without becoming infected. Nearly half of them have CTL directed towards epitopes previously defined for B clade virus, which are largely conserved in the A and D clade sequences. Stronger responses are frequently elicited using the A or D clade version of an epitope to stimulate CTL, suggesting that they were originally primed by exposure to these virus strains. CTL responses have been defined to novel epitopes presented by HLA class I molecules associated with resistance to infection in the cohort, HLA-A*6802 and HLA-B18. Estimates using a modified interferon-gamma Elispot assay indicate a circulating frequency of CTL to individual epitopes of between 1:3,200 and 1:50,000. Thus, HIV-specific immune responses-particularly cross-clade CTL activity- may be responsible for protection against persistent HIV infection in these African women. PMID:9802890

  17. Conserved B-Cell Epitopes among Human Bocavirus Species Indicate Potential Diagnostic Targets

    PubMed Central

    Wang, Yaying; Zhou, Hongli; Wu, Chao; Paranhos-Baccalà, Gláucia; Vernet, Guy; Guo, Li; Wang, Jianwei

    2014-01-01

    Background Human bocavirus species 1–4 (HBoV1–4) have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs. Methodology/Principal Findings We generated HBoV1–3 VP2 gene fragment phage display libraries (GFPDLs) and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides–P1 (1MSDTDIQDQQPDTVDAPQNT20), and P2 (162EHAYPNASHPWDEDVMPDL180)–that are conserved among HBoV1–4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1–4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4). Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide. Conclusions/Significance The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools. PMID:24475201

  18. 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.

  19. Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

    PubMed Central

    Bian, Yixin; Zhao, Shuoxian; Zhu, Shaomei; Zeng, Jinfeng; Li, Tingting; Fu, Yongshui; Wang, Yuanzhan; Zheng, Xin; Zhang, Ling; Wang, Wenjing; Yang, Baocheng; Zhou, Yuanping; Allain, Jean-Pierre; Li, Chengyao

    2013-01-01

    Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection. PMID:23894620

  20. 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

  1. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression.

    PubMed

    Miller, J B; Teal, S B; Stockdale, F E

    1989-08-05

    A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that

  2. Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

    PubMed Central

    Kim, J J; Phillips, N J; Gibson, B W; Griffiss, J M; Yamasaki, R

    1994-01-01

    Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O

  3. Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

    PubMed Central

    Wu, Xiaoying; Li, Xiaojun; Zhang, Qingshan; Wulin, Shaozhou; Bai, Xiaofei; Zhang, Tingting; Wang, Yue; Liu, Ming; Zhang, Yun

    2015-01-01

    Background The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. Methods and Results To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. Conclusions and Significance We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1. PMID:25706372

  4. 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.

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

    PubMed

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

    2015-01-01

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

  6. 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

  7. Conservation and Accessibility of an Inner Core Lipopolysaccharide Epitope of Neisseria meningitidis

    PubMed Central

    Plested, Joyce S.; Makepeace, Katherine; Jennings, Michael P.; Gidney, Margaret Anne J.; Lacelle, Suzanne; Brisson, J.-R.; Cox, Andrew D.; Martin, Adele; Bird, A. Graham; Tang, Christoph M.; Mackinnon, Fiona M.; Richards, James C.; Moxon, E. Richard

    1999-01-01

    We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the β-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis. PMID:10496924

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

    PubMed

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

    2015-02-01

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

  9. Computational approach for predicting the conserved B-cell epitopes of hemagglutinin H7 subtype influenza virus

    PubMed Central

    Wang, Xiangyu; Sun, Qi; Ye, Zhonghua; Hua, Ying; Shao, Na; Du, Yanli; Zhang, Qiwei; Wan, Chengsong

    2016-01-01

    An avian-origin influenza H7N9 virus epidemic occurred in China in 2013–2014, in which >422 infected people suffered from pneumonia, respiratory distress syndrome and septic shock. H7N9 viruses belong to the H7 subtype of avian-origin influenza viruses (AIV-H7). Hemagglutinin (HA) is a vital membrane protein of AIV that has an important role in host recognition and infection. The epitopes of HA are significant determinants of the regularity of epidemic and viral mutation and recombination mechanisms. The present study aimed to predict the conserved B-cell epitopes of AIV-H7 HA using a bioinformatics approach, including the three most effective epitope prediction softwares available online: Artificial Neural Network based B-cell Epitope Prediction (ABCpred), B-cell Epitope Prediction (BepiPred) and Linear B-cell Epitope Prediction (LBtope). A total of 24 strains of Euro-Asiatic AIV-H7 that had been associated with a serious poultry pandemic or had infected humans in the past 30 years were selected to identify the conserved regions of HA. Sequences were obtained from the National Center for Biotechnology Information and Global Initiative on Sharing Avian Influenza Data databases. Using a combination of software prediction and sequence comparisons, the conserved epitopes of AIV-H7 were predicted and clarified. A total of five conserved epitopes [amino acids (aa) 37–52, 131–142, 215–234, 465–484 and 487–505] with a suitable length, high antigenicity and minimal variation were predicted and confirmed. Each obtained a score of >0.80 in ABCpred, 60% in LBtope and a level of 0.35 in Bepipred. In addition, a representative amino acid change (glutamine235-to-leucine235) in the HA protein of the 2013 AIV-H7N9 was discovered. The strategy adopted in the present study may have profound implications on the rapid diagnosis and control of infectious disease caused by H7N9 viruses, as well as by other virulent viruses, such as the Ebola virus. PMID:27703505

  10. Identification of a gag protein epitope conserved among all four groups of primate immunodeficiency viruses by using monoclonal antibodies.

    PubMed

    Otteken, A; Nick, S; Bergter, W; Voss, G; Faisst, A C; Stahl-Hennig, C; Hunsmann, G

    1992-10-01

    Five monoclonal antibodies (MAbs) were raised against the gag proteins of simian immunodeficiency virus (SIV) from African green monkey (SIVagmTYO-7). Two MAbs reacted with the matrix protein p17 and the other three with the core protein p24. Studies on the cross-reactivity of the MAbs revealed that the anti-p24 MAbs detected an epitope shared by the viruses belonging to the human immunodeficiency virus type 2 (HIV-2)/SIVmac group and SIVagmTYO-7 and SIVagmTYO-5. The anti-p17 MAbs recognized an epitope present on all these viruses and on SIVagmTYO-1, HIV-1 and SIVmnd. This finding demonstrates for the first time that the matrix protein, p17 or p18, respectively, of all nine HIV and SIV isolates tested in this study expresses at least one conserved immunogenic epitope recognized serologically. By using synthetic peptides, this epitope was identified at the N terminus of p17. Furthermore, this epitope was analysed by multiple sequence alignments of the peptide with homologous sequences of HIV and SIV p17.

  11. Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

    PubMed Central

    Dutta, Sheetij; Dlugosz, Lisa S.; Drew, Damien R.; Ge, Xiopeng; Ababacar, Diouf; Rovira, Yazmin I.; Moch, J. Kathleen; Shi, Meng; Long, Carole A.; Foley, Michael; Beeson, James G.; Anders, Robin F.; Miura, Kazutoyo; Haynes, J. David; Batchelor, Adrian H.

    2013-01-01

    Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal

  12. Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus

    PubMed Central

    Klausberger, Miriam; Tscheliessnig, Rupert; Neff, Silke; Nachbagauer, Raffael; Wohlbold, Teddy John; Wilde, Monika; Palmberger, Dieter; Krammer, Florian; Jungbauer, Alois; Grabherr, Reingard

    2016-01-01

    Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection. PMID:27088239

  13. 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

  14. 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.

  15. 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.

  16. Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

    PubMed Central

    Ye, Xiaohua; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Shi, Jinping; Zhang, Yunfang; Kong, Liangliang; Cong, Yao

    2014-01-01

    Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies. PMID:24131712

  17. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  18. 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

  19. Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

    PubMed Central

    Guo, Li; Wu, Chao; Gonzalez, Richard; Paranhos-Baccalà, Gláucia; Vernet, Guy; Wang, Jianwei; Hung, Tao

    2011-01-01

    Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs. PMID:21886787

  20. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

    PubMed

    Grandea, Andres G; Olsen, Ole A; Cox, Thomas C; Renshaw, Mark; Hammond, Philip W; Chan-Hui, Po-Ying; Mitcham, Jennifer L; Cieplak, Witold; Stewart, Shaun M; Grantham, Michael L; Pekosz, Andrew; Kiso, Maki; Shinya, Kyoko; Hatta, Masato; Kawaoka, Yoshihiro; Moyle, Matthew

    2010-07-13

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

  1. Cytolytic T lymphocytes (CTLs) from HIV-1 subtype C-infected Indian patients recognize CTL epitopes from a conserved immunodominant region of HIV-1 Gag and Nef.

    PubMed

    Thakar, Madhuri R; Bhonge, Leena S; Lakhashe, Samir K; Shankarkumar, U; Sane, Suvarna S; Kulkarni, Smita S; Mahajan, Bharati A; Paranjape, Ramesh S

    2005-09-01

    Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.

  2. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.

    PubMed

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E; Blackwelder, William C; Emerson, Suzanne U; Cosset, François-Loïc; Purcell, Robert H

    2003-11-25

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.

  3. In vitro assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes

    PubMed Central

    Bartosch, Birke; Bukh, Jens; Meunier, Jean-Christophe; Granier, Christelle; Engle, Ronald E.; Blackwelder, William C.; Emerson, Suzanne U.; Cosset, François-Loïc; Purcell, Robert H.

    2003-01-01

    Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C. PMID:14617769

  4. 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

  5. A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A/Goose/Guangdong/1/96 hemagglutinin.

    PubMed

    Zhu, Xueyong; Guo, Yong-Hui; Jiang, Tao; Wang, Ya-Di; Chan, Kwok-Hung; Li, Xiao-Feng; Yu, Wenli; McBride, Ryan; Paulson, James C; Yuen, Kwok-Yung; Qin, Cheng-Feng; Che, Xiao-Yan; Wilson, Ian A

    2013-12-01

    Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab' fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.

  6. A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

    PubMed

    Doyle, Tracey M; Li, Changgui; Bucher, Doris J; Hashem, Anwar M; Van Domselaar, Gary; Wang, Junzhi; Farnsworth, Aaron; She, Yi-Min; Cyr, Terry; He, Runtao; Brown, Earl G; Hurt, Aeron C; Li, Xuguang

    2013-11-08

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.

  7. 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.

  8. Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library

    PubMed Central

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

    2015-01-01

    The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection. PMID:26121648

  9. Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.

    PubMed

    Seow, Jeffrey; Morales, Rodrigo A V; MacRaild, Christopher A; Krishnarjuna, Bankala; McGowan, Sheena; Dingjan, Tamir; Jaipuria, Garima; Rouet, Romain; Wilde, Karyn L; Atreya, Hanudatta S; Richards, Jack S; Anders, Robin F; Christ, Daniel; Drinkwater, Nyssa; Norton, Raymond S

    2017-03-24

    Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

  10. 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.

  11. 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.

  12. Serologic reactivity using conserved envelope epitopes in feline lentivirus-infected felids.

    PubMed

    Kania, S A; Kennedy, M A; Potgieter, L N

    1997-04-01

    An enzyme-linked immunosorbent assay (ELISA) based on synthetic peptides identical to lentivirus envelope protein amino acid sequences was used to study serologic reactivity of lentivirus-infected domestic cats and nondomestic felids. One feline immunodeficiency virus (FIV) peptide, P237, was consistently recognized by antibodies from FIV-infected cats, but 2 other FIV peptide antigens were not. The molecular basis for this serologic reactivity was examined. Lentivirus-infected nondomestic Felis species reacted intensely with a puma lentivirus (PLV) peptide corresponding to the conserved FIV peptide. However, lentivirus-infected Panthera species, from which a different lentivirus has been isolated, did not react with the PLV. FIV-infected domestic felids also did not have significant reactivity with the PLV peptide. The peptide ELISA is comparable in sensitivity and specificity to western blot analysis and a commercial enzyme immunoassay. Unlike the other assays, however, the peptide ELISA is inexpensive, requires a small amount of serum, enables the study of specific isotype reactivity, and discriminates between antibodies to FIV and those to PLV. Antibody tests based upon the FIV and the PLV peptides should be useful for detecting the possible introduction of FIV into exotic felids or of lentiviruses from nondomestic felids into the domestic cat population.

  13. Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

    PubMed Central

    Ahmed, Tina; Borthwick, Nicola J.; Gilmour, Jill; Hayes, Peter; Dorrell, Lucy; Hanke, Tomáš

    2016-01-01

    Objective The specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. Design CD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. Methods Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. Results We formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. Conclusions These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient

  14. Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

    PubMed

    Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

    2015-11-18

    Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection.

  15. Characterizing the interactions between a naturally primed immunoglobulin A and its conserved Bacteroides thetaiotaomicron species-specific epitope in gnotobiotic mice.

    PubMed

    Peterson, Daniel A; Planer, Joseph D; Guruge, Janaki L; Xue, Lai; Downey-Virgin, Whitt; Goodman, Andrew L; Seedorf, Henning; Gordon, Jeffrey I

    2015-05-15

    The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1-/-, or Myd88-/- mice. Comparison of gnotobiotic Rag1-/- mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism.

  16. Alternative Recognition of the Conserved Stem Epitope in Influenza A Virus Hemagglutinin by a VH3-30-Encoded Heterosubtypic Antibody

    PubMed Central

    Wyrzucki, Arkadiusz; Dreyfus, Cyrille; Kohler, Ines; Steck, Marco

    2014-01-01

    ABSTRACT A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes. IMPORTANCE Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic

  17. Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

    PubMed

    Wawegama, Nadeeka K; Tarigan, Simson; Indriani, Risa; Selleck, Paul; Adjid, Rm Abdul; Syafriati, Tati; Hardiman; Durr, Peter A; Ignjatovic, Jagoda

    2016-08-01

    A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

  18. Molecular and Genetic Characterization of HIV-1 Tat Exon-1 Gene from Cameroon Shows Conserved Tat HLA-Binding Epitopes: Functional Implications

    PubMed Central

    Teto, Georges; Fonsah, Julius Y.; Tagny, Claude T.; Mbanya, Dora; Nchindap, Emilienne; Kenmogne, Leopoldine; Fokam, Joseph; Njamnshi, Dora M.; Kouanfack, Charles; Njamnshi, Alfred K.; Kanmogne, Georgette D.

    2016-01-01

    HIV-1 Tat plays a critical role in viral transactivation. Subtype-B Tat has potential use as a therapeutic vaccine. However, viral genetic diversity and population genetics would significantly impact the efficacy of such a vaccine. Over 70% of the 37-million HIV-infected individuals are in sub-Saharan Africa (SSA) and harbor non-subtype-B HIV-1. Using specimens from 100 HIV-infected Cameroonians, we analyzed the sequences of HIV-1 Tat exon-1, its functional domains, post-translational modifications (PTMs), and human leukocyte antigens (HLA)-binding epitopes. Molecular phylogeny revealed a high genetic diversity with nine subtypes, CRF22_01A1/CRF01_AE, and negative selection in all subtypes. Amino acid mutations in Tat functional domains included N24K (44%), N29K (58%), and N40K (30%) in CRF02_AG, and N24K in all G subtypes. Motifs and phosphorylation analyses showed conserved amidation, N-myristoylation, casein kinase-2 (CK2), serine and threonine phosphorylation sites. Analysis of HLA allelic frequencies showed that epitopes for HLAs A*0205, B*5301, Cw*0401, Cw*0602, and Cw*0702 were conserved in 58%–100% of samples, with B*5301 epitopes having binding affinity scores > 100 in all subtypes. This is the first report of N-myristoylation, amidation, and CK2 sites in Tat; these PTMs and mutations could affect Tat function. HLA epitopes identified could be useful for designing Tat-based vaccines for highly diverse HIV-1 populations, as in SSA. PMID:27438849

  19. V1/V2 Neutralizing Epitope is Conserved in Divergent Non-M Groups of HIV-1

    PubMed Central

    Morgand, Marion; Bouvin-Pley, Mélanie; Plantier, Jean-Christophe; Moreau, Alain; Alessandri, Elodie; Simon, François; Pace, Craig S.; Pancera, Marie; Ho, David D.; Poignard, Pascal; Bjorkman, Pamela J.; Mouquet, Hugo; Nussenzweig, Michel C.; Kwong, Peter D.; Baty, Daniel; Chames, Patrick; Braibant, Martine

    2016-01-01

    Background: Highly potent broadly neutralizing monoclonal antibodies (bNAbs) have been obtained from individuals infected by HIV-1 group M variants. We analyzed the cross-group neutralization potency of these bNAbs toward non-M primary isolates (PI). Material and Methods: The sensitivity to neutralization was analyzed in a neutralization assay using TZM-bl cells. Twenty-three bNAbs were used, including reagents targeting the CD4-binding site, the N160 glycan-V1/V2 site, the N332 glycan-V3 site, the membrane proximal external region of gp41, and complex epitopes spanning both env subunits. Two bispecific antibodies that combine the inhibitory activity of an anti-CD4 with that of PG9 or PG16 bNAbs were included in the study (PG9-iMab and PG16-iMab). Results: Cross-group neutralization was observed only with the bNAbs targeting the N160 glycan-V1/V2 site. Four group O PIs, 1 group N PI, and the group P PI were neutralized by PG9 and/or PG16 or PGT145 at low concentrations (0.04–9.39 μg/mL). None of the non-M PIs was neutralized by the bNAbs targeting other regions at the highest concentration tested, except 10E8 that neutralized weakly 2 group N PIs and 35O22 that neutralized 1 group O PI. The bispecific bNAbs neutralized very efficiently all the non-M PIs with IC50 below 1 μg/mL, except 2 group O strains. Conclusion: The N160 glycan-V1/V2 site is the most conserved neutralizing site within the 4 groups of HIV-1. This makes it an interesting target for the development of HIV vaccine immunogens. The corresponding bNAbs may be useful for immunotherapeutic strategies in patients infected by non-M variants. PMID:26413851

  20. Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display

    PubMed Central

    Crossey, Erin; Frietze, Kathryn; Narum, David L.; Peabody, David S.; Chackerian, Bryce

    2015-01-01

    We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the Plasmodium falciparum blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach. PMID:26147502

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

    PubMed

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

    2009-12-30

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

  2. 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

  3. Generation of monoclonal antibodies reactive against subtype specific conserved B-cell epitopes on haemagglutinin protein of influenza virus H5N1.

    PubMed

    Fiebig, Petra; Shehata, Awad A; Liebert, Uwe G

    2015-03-02

    H5-specific monoclonal antibodies may serve as valuable tools for rapid diagnosis of H5N1 avian influenza virus. Therefore, conserved H5-specific sequences of the haemagglutinin (HA) protein were expressed in Pichia pastoris and used for generation of monoclonal antibodies (mAbs). The two mAbs, FD6 and HE4, were strongly reactive against native HA protein and exhibited specificity for subtype H5. By epitope mapping linear epitopes of mAbs were identified that are highly conserved among influenza A virus of subtype H5. Additionally no sequence similarities to homologous regions on HA proteins of other influenza A virus subtypes (i.e. H1, H3, H7, H9) were detected by sequence alignment analysis. Both mAbs did not cross react with native or denatured HA proteins of other influenza A virus subtypes. Furthermore, using ELISA and immunofluorescence test mAb FD6 reacted only to the native H5 protein of recently circulating highly pathogenic H5N1 influenza viruses but not to low pathogenic H5N1 isolates. In conclusion, the use of the two mAbs in non-molecular tests like antigen-capture-ELISA appears promising for detecting influenza A H5N1 virus.

  4. Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    PubMed Central

    Wen, Xuexia; Bao, Hongmei; Shi, Lin; Tao, Qimeng; Jiang, Yongping; Zeng, Xianying; Xu, Xiaolong; Tian, Guobin; Zheng, Shimin; Chen, Hualan

    2016-01-01

    Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza. PMID:26938453

  5. Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. Results In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. Conclusion The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex. PMID:21375771

  6. 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

  7. 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.

  8. The N-terminus of the Montano virus nucleocapsid protein possesses broadly cross-reactive conformation-dependent epitopes conserved in rodent-borne hantaviruses.

    PubMed

    Saasa, Ngonda; Yoshida, Haruka; Shimizu, Kenta; Sánchez-Hernández, Cornelio; Romero-Almaraz, María de Lourdes; Koma, Takaaki; Sanada, Takahiro; Seto, Takahiro; Yoshii, Kentaro; Ramos, Celso; Yoshimatsu, Kumiko; Arikawa, Jiro; Takashima, Ikuo; Kariwa, Hiroaki

    2012-06-20

    The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.

  9. Computational analysis of perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

    PubMed Central

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV) in three countries, provides key insights in developing strategies for tackling ZIKV, which has caused global panic to microcephaly and Guillain-Barre Syndrome. Dengue virus (DENV), a member of the family Flaviviridae, the causal agent of the self-limiting Dengue fever and the potentially fatal hemorrhagic fever/dengue shock syndrome, has been a scourge in tropical countries for many centuries. The recently solved structure of mature ZIKV (PDB ID:5IRE) has provided key insights into the structure of the envelope (E) and membrane (M) proteins, the primary target of neutralizing antibodies. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes, and also enumerates residue pairs that undergo significant polarity reversal. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little

  10. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

    NASA Technical Reports Server (NTRS)

    Lim, Kap; Ho, Joseph X.; Keeling, Kim; Gilliland, Gary L.; Ji, Xinhua; Rueker, Florian; Carter, Daniel C.

    1994-01-01

    The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(sub 3)2(sub 1)2 with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

  11. Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup.

    PubMed

    Chen, Tsung-Chi; Huang, Ching-Wen; Kuo, Yan-Wen; Liu, Fang-Lin; Yuan, Chao-Hsiu Hsuan; Hsu, Hei-Ti; Yeh, Shyi-Dong

    2006-12-01

    ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.

  12. Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

    PubMed Central

    Xue, Mei; Shi, Xingming; Zhang, Jing; Zhao, Yan; Cui, Hongyu; Hu, Shunlei; Gao, Hongbo; Cui, Xianlan; Wang, Yun-Feng

    2012-01-01

    Background The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. Methods and Results This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched 213SVQYHPL219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that 213SVQYHPL219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. Conclusions and Significance We identified 213SVQYHPL219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. PMID:23185456

  13. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

    PubMed Central

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Rüker, F.; Carter, D. C.

    1994-01-01

    The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed. PMID:7538846

  14. Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

    NASA Technical Reports Server (NTRS)

    Lim, K.; Ho, J. X.; Keeling, K.; Gilliland, G. L.; Ji, X.; Ruker, F.; Carter, D. C.

    1994-01-01

    The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

  15. Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope.

    PubMed

    Chen, Shaoheng; Zheng, Dan; Li, Changgui; Zhang, Wenjie; Xu, Wenting; Liu, Xueying; Fang, Fang; Chen, Ze

    2015-01-01

    We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB(*)) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB(*) adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

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

    PubMed Central

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

    2016-01-01

    Background Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. Methods and Results To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. Conclusions and Significance We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV. PMID:27191594

  17. The Human CD8+ T Cell Responses Induced by a Live Attenuated Tetravalent Dengue Vaccine Are Directed against Highly Conserved Epitopes

    PubMed Central

    Angelo, Michael A.; Bangs, Derek J.; Sidney, John; Paul, Sinu; Peters, Bjoern; de Silva, Aruna D.; Lindow, Janet C.; Diehl, Sean A.; Whitehead, Stephen; Durbin, Anna; Kirkpatrick, Beth; Sette, Alessandro

    2014-01-01

    ABSTRACT The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV. IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV

  18. Conservation of Babesia bovis small heat shock protein (Hsp20) among strains and definition of T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes.

    PubMed

    Norimine, Junzo; Mosqueda, Juan; Palmer, Guy H; Lewin, Harris A; Brown, Wendy C

    2004-02-01

    Babesia bovis small heat shock protein (Hsp20) is recognized by CD4+ T lymphocytes from cattle that have recovered from infection and are immune to challenge. This candidate vaccine antigen is related to a protective antigen of Toxoplasma gondii, Hsp30/bag1, and both are members of the alpha-crystallin family of proteins that can serve as molecular chaperones. In the present study, immunofluorescence microscopy determined that Hsp20 is expressed intracellularly in all merozoites. Importantly, Hsp20 is also expressed by tick larval stages, including sporozoites, so that natural tick-transmitted infection could boost a vaccine-induced response. The predicted amino acid sequence of Hsp20 from merozoites is completely conserved among different B. bovis strains. To define the location of CD4+ T-cell epitopes for inclusion in a multiepitope peptide or minigene vaccine construct, truncated recombinant Hsp20 proteins and overlapping peptides were tested for their ability to stimulate T cells from immune cattle. Both amino-terminal (amino acids [aa] 1 to 105) and carboxy-terminal (aa 48 to 177) regions were immunogenic for the majority of cattle in the study, stimulating strong proliferation and IFN-gamma production. T-cell lines from all individuals with distinct DRB3 haplotypes responded to aa 11 to 62 of Hsp20, which contained one or more immunodominant epitopes for each animal. One epitope, DEQTGLPIKS (aa 17 to 26), was identified by T-cell clones. The presence of strain-conserved T helper cell epitopes in aa 11 to 62 of the ubiquitously expressed Hsp20 that are presented by major histocompatibility complex class II molecules represented broadly in the Holstein breed supports the inclusion of this region in vaccine constructs to be tested in cattle.

  19. Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (PvCelTOS) gene sequence and potential epitopes are highly conserved among isolates from different regions of Brazilian Amazon

    PubMed Central

    Bitencourt Chaves, Lana; Perce-da-Silva, Daiana de Souza; Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Cassiano, Gustavo Capatti; Machado, Ricardo Luiz Dantas; Pratt-Riccio, Lilian Rose; Banic, Dalma Maria

    2017-01-01

    The Plasmodium vivax Cell-traversal protein for ookinetes and sporozoites (PvCelTOS) plays an important role in the traversal of host cells. Although essential to PvCelTOS progress as a vaccine candidate, its genetic diversity remains uncharted. Therefore, we investigated the PvCelTOS genetic polymorphism in 119 field isolates from five different regions of Brazilian Amazon (Manaus, Novo Repartimento, Porto Velho, Plácido de Castro and Oiapoque). Moreover, we also evaluated the potential impact of non-synonymous mutations found in the predicted structure and epitopes of PvCelTOS. The field isolates showed high similarity (99.3% of bp) with the reference Sal-1 strain, presenting only four Single-Nucleotide Polymorphisms (SNP) at positions 24A, 28A, 109A and 352C. The frequency of synonymous C109A (82%) was higher than all others (p<0.0001). However, the non-synonymous G28A and G352C were observed in 9.2% and 11.7% isolates. The great majority of the isolates (79.8%) revealed complete amino acid sequence homology with Sal-1, 10.9% presented complete homology with Brazil I and two undescribed PvCelTOS sequences were observed in 9.2% field isolates. Concerning the prediction analysis, the N-terminal substitution (Gly10Ser) was predicted to be within a B-cell epitope (PvCelTOS Accession Nos. AB194053.1) and exposed at the protein surface, while the Val118Leu substitution was not a predicted epitope. Therefore, our data suggest that although G28A SNP might interfere in potential B-cell epitopes at PvCelTOS N-terminal region the gene sequence is highly conserved among the isolates from different geographic regions, which is an important feature to be taken into account when evaluating its potential as a vaccine candidate. PMID:28158176

  20. In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Merozoite Surface Antigens-2 of Babesia bovis conform a family of GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes, thus constituting putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-...

  1. CD4+ T cell epitopes of FliC conserved between strains of Burkholderia - implications for vaccines against melioidosis and Cepacia Complex in Cystic Fibrosis

    PubMed Central

    Musson, Julie A.; Reynolds, Catherine J; Rinchai, Darawan; Nithichanon, Arnone; Khaenam, Prasong; Favry, Emmanuel; Spink, Natasha; Chu, Karen KY; De Soyza, Anthony; Bancroft, Gregory J; Lertmemongkolchai, Ganjana; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M.; Robinson, John H.

    2014-01-01

    Burkholderia pseudomallei (Bp), is the causative agent of melioidosis, characterized by pneumonia and fatal septicemia and prevalent in SE Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The Bp flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed Bp FliC peptide binding affinity to multiple HLA class II alleles, then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between Bp and the related Burkholderia species associated with Cepacia Complex CF. Bp FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in Bp-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic Bp FliC epitope also cross-reacted with orthologous FliC sequences from B. multivorans and B. cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II antigen presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in Bp endemic regions as well as CF patients. PMID:25392525

  2. CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

    PubMed

    Musson, Julie A; Reynolds, Catherine J; Rinchai, Darawan; Nithichanon, Arnone; Khaenam, Prasong; Favry, Emmanuel; Spink, Natasha; Chu, Karen K Y; De Soyza, Anthony; Bancroft, Gregory J; Lertmemongkolchai, Ganjana; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M; Robinson, John H

    2014-12-15

    Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.

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

    PubMed Central

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

    2017-01-01

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

  4. [Detection of conservative and variable epitopes of the pandemic influenza virus A(H1N1)pdm09 hemagglutinin using monoclonal antibodies].

    PubMed

    Masalova, O V; Chichev, E V; Fediakina, I T; Mukasheva, E A; Klimova, R R; Shchelkanov, M Iu; Burtseva, E I; Ivanova, V T; Kushch, A A; L'vov, D K

    2014-01-01

    The goal of this work was to analyze the antigenic structure of the hemagglutinin (HA) of the pandemic influenza virus A(H1N1)pdm09 using monoclonal antibodies (MAbs) and to develop a sandwich ELISA for identification of pandemic strains. Competitive ELISA demonstrated that 6 MAbs against HA of the pandemic influenza A/ IIV-Moscow/01/2009 (H1N1)pdm09 virus identified six epitopes. Binding of MAbs with 22 strains circulating in Russian Federation during 2009-2012 was analyzed in the hemagglutination-inhibition test (HI). The MAbs differed considerably in their ability to decrease the HI activity of these strains. MAb 5F7 identified all examined strains; MAbs 3A3 and 10G2 reacted with the majority of them. A highly sensitive sandwich ELISA was constructed based on these three MAbs that can differentiate the pandemic influenza strains from the seasonal influenza virus. The constancy of the HA epitope that reacts with MAb 5F7 provides its use for identification of the pandemic influenza strains in HI test. MAbs 3D9, 6A3 and 1E7 are directed against the variable HA epitopes, being sensitive to several amino acid changes in Sa, Sb, and Ca2 antigenic sites and in receptor binding site. These MAbs can be used to detect differences in HA structure and to study the antigenic drift of the pandemic influenza virus A(H1N1)pdm09.

  5. Conservation of G-Protein Epitopes in Respiratory Syncytial Virus (Group A) Despite Broad Genetic Diversity: Is Antibody Selection Involved in Virus Evolution?

    PubMed Central

    Trento, Alfonsina; Ábrego, Leyda; Rodriguez-Fernandez, Rosa; González-Sánchez, Maria Isabel; González-Martínez, Felipe; Delfraro, Adriana; Pascale, Juan M.; Arbiza, Juan

    2015-01-01

    ABSTRACT Worldwide G-glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than 50 years of recorded history. Multiple genotypes cocirculated during prolonged periods of time, but recent dominance of the GA2 genotype was noticed in several studies, and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with monoclonal antibodies (MAbs) that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated with the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as a major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV G-protein variability. IMPORTANCE An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of nonsynonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS values at certain sites predictive of positive selection. Since these sites cluster preferentially in the C-terminal third of the G protein, like certain epitopes recognized by murine antibodies, it was proposed that immune (antibody) selection might be driving the apparent positive selection, analogous to the antigenic drift observed in the influenza virus hemagglutinin (HA). However, careful antigenic and genetic comparison of the G glycoprotein does not provide evidence of antigenic drift in the G molecule, in agreement with recently published data which did not indicate antigenic drift in the G protein with human sera. Alternative explanations to the immune-driven selection

  6. 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.

  7. Conservation.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  8. 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.

  9. 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

  10. 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.

  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

    epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines. PMID:23584251

  12. 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

  13. In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential.

    PubMed

    Muñoz-Medina, José Esteban; Sánchez-Vallejo, Carlos Javier; Méndez-Tenorio, Alfonso; Monroy-Muñoz, Irma Eloísa; Angeles-Martínez, Javier; Santos Coy-Arechavaleta, Andrea; Santacruz-Tinoco, Clara Esperanza; González-Ibarra, Joaquín; Anguiano-Hernández, Yu-Mei; González-Bonilla, César Raúl; Ramón-Gallegos, Eva; Díaz-Quiñonez, José Alberto

    2015-01-01

    The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques.

  14. In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential

    PubMed Central

    Muñoz-Medina, José Esteban; Sánchez-Vallejo, Carlos Javier; Méndez-Tenorio, Alfonso; Monroy-Muñoz, Irma Eloísa; Angeles-Martínez, Javier; Santos Coy-Arechavaleta, Andrea; Santacruz-Tinoco, Clara Esperanza; González-Ibarra, Joaquín; Anguiano-Hernández, Yu-Mei; González-Bonilla, César Raúl; Ramón-Gallegos, Eva; Díaz-Quiñonez, José Alberto

    2015-01-01

    The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques. PMID:26346523

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  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. 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

  7. Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

    PubMed

    Piazza, P; Campbell, D; Marques, E; Hildebrand, W H; Buchli, R; Mailliard, R; Rinaldo, C R

    2014-09-01

    Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

  8. 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

  9. Expressing Redundancy among Linear-Epitope Sequence Data Based on Residue-Level Physicochemical Similarity in the Context of Antigenic Cross-Reaction

    PubMed Central

    2016-01-01

    Epitope-based design of vaccines, immunotherapeutics, and immunodiagnostics is complicated by structural changes that radically alter immunological outcomes. This is obscured by expressing redundancy among linear-epitope data as fractional sequence-alignment identity, which fails to account for potentially drastic loss of binding affinity due to single-residue substitutions even where these might be considered conservative in the context of classical sequence analysis. From the perspective of immune function based on molecular recognition of epitopes, functional redundancy of epitope data (FRED) thus may be defined in a biologically more meaningful way based on residue-level physicochemical similarity in the context of antigenic cross-reaction, with functional similarity between epitopes expressed as the Shannon information entropy for differential epitope binding. Such similarity may be estimated in terms of structural differences between an immunogen epitope and an antigen epitope with reference to an idealized binding site of high complementarity to the immunogen epitope, by analogy between protein folding and ligand-receptor binding; but this underestimates potential for cross-reactivity, suggesting that epitope-binding site complementarity is typically suboptimal as regards immunologic specificity. The apparently suboptimal complementarity may reflect a tradeoff to attain optimal immune function that favors generation of immune-system components each having potential for cross-reactivity with a variety of epitopes. PMID:27274725

  10. 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

  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. 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.

  14. 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

  15. 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

  16. M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens.

    PubMed

    Coscolla, Mireia; Copin, Richard; Sutherland, Jayne; Gehre, Florian; de Jong, Bouke; Owolabi, Olumuiya; Mbayo, Georgetta; Giardina, Federica; Ernst, Joel D; Gagneux, Sebastien

    2015-11-11

    Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.

  17. 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.

  18. Conservation of the lipooligosaccharide synthesis locus lgt among strains of Neisseria gonorrhoeae: requirement for lgtE in synthesis of the 2C7 epitope and of the beta chain of strain 15253

    PubMed Central

    1996-01-01

    The present study was undertaken to examine the extent to which the lgt locus varies among strains of gonococci. This locus encodes five glycosyl transferases involved in the synthesis of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae. We examined seven gonococcal strains and found that the structure of the lgt locus is conserved among six of these strains. The locus is strikingly altered in strain 15253. This is one of the few strains where extensive structural analysis of its LOS is available, and therefore, we defined the altered lgt locus and focused on the reactivity of mAB 2C7. We found that strain 15253 contains only two lgt genes, lgtA and lgtE. As in F62, lgtA encodes a GlcNAc transferase and is subject to phase variation. In addition, by analysis of deletion mutants, we found that lgtE, which encodes a galactosyl transferase that is required for elongating the alpha-chain, is also necessary for completing the beta chain. PMID:8879194

  19. 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.

  20. 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

  1. Molecular construction of HIV-gp120 discontinuous epitope mimics by assembly of cyclic peptides on an orthogonal alkyne functionalized TAC-scaffold.

    PubMed

    Werkhoven, P R; Elwakiel, M; Meuleman, T J; Quarles van Ufford, H C; Kruijtzer, J A W; Liskamp, R M J

    2016-01-14

    Mimics of discontinuous epitopes of for example bacterial or viral proteins may have considerable potential for the development of synthetic vaccines, especially if conserved epitopes can be mimicked. However, due to the structural complexity and size of discontinuous epitopes molecular construction of these mimics remains challeging. We present here a convergent route for the assembly of discontinuous epitope mimics by successive azide alkyne cycloaddition on an orthogonal alkyne functionalized scaffold. Here the synthesis of mimics of the HIV gp120 discontinuous epitope that interacts with the CD4 receptor is described. The resulting protein mimics are capable of inhibition of the gp120-CD4 interaction. The route is convergent, robust and should be applicable to other discontinuous epitopes.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates.

    PubMed Central

    Marcom, K A; Pearson, L D; Chung, C S; Poulson, J M; DeMartini, J C

    1991-01-01

    Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats. Images PMID:1715884

  9. Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein.

    PubMed

    Nadugala, Mahesha N; Premaratne, Prasad H; Goonasekara, Charitha L

    2016-01-01

    B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48-55), epitope number 4 of E protein at amino acids 48-55, EP9/E (165-182), EP11/E (218-233), EP20/E (322-349), EP21/E (326-353), EP23/E (356-365), and EP25/E (380-386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30-41) located in domain-I and EP26/E (393-409), EP27/E (416-435), EP28/E (417-430) located in the stem region of E protein, and EP8/prM (93-112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16-21), EP5/E (62-123), EP6/E (63-89), EP19/E (310-329), and EP24/E (371-402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes.

  10. Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein

    PubMed Central

    Nadugala, Mahesha N.

    2016-01-01

    B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48–55), epitope number 4 of E protein at amino acids 48–55, EP9/E (165–182), EP11/E (218–233), EP20/E (322–349), EP21/E (326–353), EP23/E (356–365), and EP25/E (380–386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30–41) located in domain-I and EP26/E (393–409), EP27/E (416–435), EP28/E (417–430) located in the stem region of E protein, and EP8/prM (93–112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16–21), EP5/E (62–123), EP6/E (63–89), EP19/E (310–329), and EP24/E (371–402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes. PMID:27688753

  11. 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.

  12. Immunogenic Consensus Sequence T helper Epitopes for a Pan-Burkholderia Biodefense Vaccine

    PubMed Central

    De Groot, Anne S.; Ardito, Matthew; Moise, Leonard; Gustafson, Eric A.; Spero, Denice; Tejada, Gloria; Martin, William

    2014-01-01

    Background Biodefense vaccines against Category B bioterror agents Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM) are needed, as they are both easily accessible to terrorists and have strong weaponization potential. Burkholderia cepaciae (BC), a related pathogen, causes chronic lung infections in cystic fibrosis patients. Since BPM, BM and BC are all intracellular bacteria, they are excellent targets for T cell-based vaccines. However, the sheer volume of available genomic data requires the aid of immunoinformatics for vaccine design. Using EpiMatrix, ClustiMer and EpiAssembler, a set of immunoinformatic vaccine design tools, we screened the 31 available Burkholderia genomes and performed initial tests of our selections that are candidates for an epitope-based multi-pathogen vaccine against Burkholderia species. Results Immunoinformatics analysis of 31 Burkholderia genomes yielded 350,004 9-mer candidate vaccine peptides of which 133,469 had perfect conservation across the 10 BM genomes, 175,722 had perfect conservation across the 11 BPM genomes and 40,813 had perfect conservation across the 10 BC genomes. Further screening with EpiMatrix yielded 54,010 high-scoring Class II epitopes; these were assembled into 2,880 longer highly conserved ‘immunogenic consensus sequence’ T helper epitopes. 100% of the peptides bound to at least one HLA class II allele in vitro, 92.7% bound to at least two alleles, 82.9% to three, and 75.6% of the binding results were consistent with the immunoinformatics analysis. Conclusions Our results show it is possible to rapidly identify promiscuous T helper epitopes conserved across multiple Burkholderia species and test their binding to HLA ligands in vitro. The next step in our process will be to test the epitopes ex vivo using peripheral leukocytes from BC, BPM infected humans and for immunogenicity in human HLA transgenic mice. We expect that this approach will lead to development of a licensable, pan

  13. PEPVAC: a web server for multi-epitope vaccine development based on the prediction of supertypic MHC ligands.

    PubMed

    Reche, Pedro A; Reinherz, Ellis L

    2005-07-01

    Prediction of peptide binding to major histocompatibility complex (MHC) molecules is a basis for anticipating T-cell epitopes, as well as epitope discovery-driven vaccine development. In the human, MHC molecules are known as human leukocyte antigens (HLAs) and are extremely polymorphic. HLA polymorphism is the basis of differential peptide binding, until now limiting the practical use of current epitope-prediction tools for vaccine development. Here, we describe a web server, PEPVAC (Promiscuous EPitope-based VACcine), optimized for the formulation of multi-epitope vaccines with broad population coverage. This optimization is accomplished through the prediction of peptides that bind to several HLA molecules with similar peptide-binding specificity (supertypes). Specifically, we offer the possibility of identifying promiscuous peptide binders to five distinct HLA class I supertypes (A2, A3, B7, A24 and B15). We estimated the phenotypic population frequency of these supertypes to be 95%, regardless of ethnicity. Targeting these supertypes for promiscuous peptide-binding predictions results in a limited number of potential epitopes without compromising the population coverage required for practical vaccine design considerations. PEPVAC can also identify conserved MHC ligands, as well as those with a C-terminus resulting from proteasomal cleavage. The combination of these features with the prediction of promiscuous HLA class I ligands further limits the number of potential epitopes. The PEPVAC server is hosted by the Dana-Farber Cancer Institute at the site http://immunax.dfci.harvard.edu/PEPVAC/.

  14. 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.

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

    PubMed Central

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

    2016-01-01

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

  16. 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.

  17. 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

  18. Identification of a linear B-cell epitope on the avian leukosis virus P27 protein using monoclonal antibodies.

    PubMed

    Li, Xiaofei; Qin, Liting; Zhu, Haibo; Sun, Yingjun; Cui, Xuezhi; Gao, Yadong; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2016-10-01

    Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein.

  19. 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.

  20. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    PubMed Central

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  1. 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

  2. Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

    PubMed

    Martyushev, Alexey P; Petravic, Janka; Grimm, Andrew J; Alinejad-Rokny, Hamid; Gooneratne, Shayarana L; Reece, Jeanette C; Cromer, Deborah; Kent, Stephen J; Davenport, Miles P

    2015-05-01

    CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

  3. A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice.

    PubMed

    Jiang, Liping; Fan, Rongjun; Sun, Shiyang; Fan, Peihu; Su, Weiheng; Zhou, Yan; Gao, Feng; Xu, Fei; Kong, Wei; Jiang, Chunlai

    2015-11-27

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.

  4. Identification of a New Broadly Cross-reactive Epitope within Domain III of the Duck Tembusu Virus E Protein

    PubMed Central

    Li, Chenxi; Bai, Xiaofei; Meng, Runze; Shaozhou, Wulin; Zhang, Qingshan; Hua, Ronghong; Liu, Jyung-Hurng; Liu, Ming; Zhang, Yun

    2016-01-01

    In 2010, a pathogenic flavivirus termed duck Tembusu virus (DTMUV) caused widespread outbreak of egg-drop syndrome in domesticated ducks in China. Although the glycoprotein E of DTMUV is an important structural component of the virus, the B-cell epitopes of this protein remains uncharacterized. Using phage display and mutagenesis, we identified a minimal B-cell epitope, 374EXE/DPPFG380, that mediates binding to a nonneutralizing monoclonal antibody. DTMUV-positive duck serum reacted with the epitope, and amino acid substitutions revealed the specific amino acids that are essential for antibody binding. Dot-blot assays of various flavivirus-positive sera indicated that EXE/DPPFG is a cross-reactive epitope in most flaviviruses, including Zika, West Nile, Yellow fever, dengue, and Japanese encephalitis viruses. These findings indicate that the epitope sequence is conserved among many strains of mosquito-borne flavivirus. Protein structure modeling revealed that the epitope is located in domain III of the DTMUV E protein. Together, these results provide new insights on the broad cross-reactivity of a B-cell binding site of the E protein of flaviviruses, which can be exploited as a diagnostic or therapeutic target for identifying, studying, or treating DTMUV and other flavivirus infections. PMID:27824100

  5. Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

    PubMed Central

    Sun, EnCheng; Zhao, Jing; Sun, Liang; Xu, QingYuan; Yang, Tao; Qin, YongLi; Wang, WenShi; Wei, Peng; Sun, Jing; Wu, DongLai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein. PMID:23922704

  6. Comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus E2 protein recognized by avian antibody responses.

    PubMed

    Sun, Encheng; Zhao, Jing; Sun, Liang; Xu, Qingyuan; Yang, Tao; Qin, Yongli; Wang, Wenshi; Wei, Peng; Sun, Jing; Wu, Donglai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211-226 and 331-352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11-26, 30-45 and 151-166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.

  7. Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein.

    PubMed

    Parra-López, Carlos; Calvo-Calle, J Mauricio; Cameron, Thomas O; Vargas, Luis E; Salazar, Luz Mary; Patarroyo, Manuel E; Nardin, Elizabeth; Stern, Lawrence J

    2006-05-26

    A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

  8. Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein.

    PubMed

    Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Tong, Mingwei; Wang, Jianke; Zhao, Hang; Lin, Peng; Cheng, Shipeng

    2016-02-02

    Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses.

  9. 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.

  10. Combination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis

    PubMed Central

    e Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals. PMID:27621732

  11. 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

  12. Identification and characterization of a cross-neutralization epitope of Enterovirus 71.

    PubMed

    Liu, Chia-Chyi; Chou, Ai-Hsiang; Lien, Shu-Pei; Lin, Hsiao-Yu; Liu, Shih-Jen; Chang, Jui-Yuan; Guo, Meng-Shin; Chow, Yen-Hung; Yang, Wun-Syue; Chang, Kate Hsuen-Wen; Sia, Charles; Chong, Pele

    2011-06-10

    , a dose-dependent relationship between the number of VP2-28 epitope units measured by a quantitative assay in vaccine preparations and the magnitude of neutralizing titers was demonstrated. VP2-28 has amino acid sequences that are highly conserved among EV71 genotypes, is not affected by formalin-treatment and long-term storage. Thus, VP2-28 could be used as the surrogate biomarker in the potency testing of candidate EV71 vaccines.

  13. Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae

    PubMed Central

    Roberts, Joanna M.; Shaw, Richard J.; Sutherland, Ian A.; Pernthaner, Anton

    2016-01-01

    Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species. PMID:27579674

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

    PubMed Central

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

    2015-01-01

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

  15. Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

    PubMed Central

    Scalia, Carla Rossana; Gendusa, Rossella; Basciu, Maria; Riva, Lorella; Tusa, Lorenza; Musarò, Antonella; Veronese, Silvio; Formenti, Angelo; D’Angelo, Donatella; Ronzio, Angela Gabriella; Bolognesi, Maddalena Maria

    2015-01-01

    The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity. PMID:26209082

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

    PubMed

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

    2015-09-25

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

  17. Epitope recognition in the human-pig comparison model on fixed and embedded material.

    PubMed

    Scalia, Carla Rossana; Gendusa, Rossella; Basciu, Maria; Riva, Lorella; Tusa, Lorenza; Musarò, Antonella; Veronese, Silvio; Formenti, Angelo; D'Angelo, Donatella; Ronzio, Angela Gabriella; Cattoretti, Giorgio; Bolognesi, Maddalena Maria

    2015-10-01

    The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig-the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Discrimination and Variable Impact of ANCA Binding to Different Surface Epitopes on Proteinase 3, the Wegener’s Autoantigen

    PubMed Central

    Silva, Francisco; Hummel, Amber M.; Jenne, Dieter E.; Specks, Ulrich

    2010-01-01

    Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener’s granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1–5/Mh1–5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system. PMID:20810247

  4. Discrimination and variable impact of ANCA binding to different surface epitopes on proteinase 3, the Wegener's autoantigen.

    PubMed

    Silva, Francisco; Hummel, Amber M; Jenne, Dieter E; Specks, Ulrich

    2010-12-01

    Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener's granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1-5/Mh1-5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system.

  5. 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

  6. 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.

  7. 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.

  8. Identification of B-cell Epitope of Leishmania donovani and its application in diagnosis of visceral leishmaniasis.

    PubMed

    Jamal, Fauzia; Dikhit, Manas Ranjan; Singh, Manish K; Shivam, Pushkar; Kumari, Sarita; Pushpanjali, Sinha; Dubey, Amit K; Kumar, Prakash; Narayan, Shyam; Gupta, Anil K; Pandey, Krishna; Das, V N R; Bimal, Sanjiva; Das, Pradeep; Singh, Shubhankar K

    2016-12-26

    Diagnosis of visceral leishmaniasis (VL) is often hindered by cross-reactions with antigens from other related parasite infections. This study aimed to develop an immunochromatographic test (ICT) which can detect the antigen present in circulating immune complexes (CICs) of VL patients using B-cell epitope-specific antibodies. MS analysis of six immunoreactive 2DE spots revealed two epitopes i.e. RFFVQGDGIGQHSLQEALERR (P1) and RRVAVLVLLDRL (P2) (From a hypothetical protein [Acc No: XP_003861458.1]). The epitope conservancy analysis suggested that the linear epitope (P1P2) is 97-100% conserved among Leishmania species and diverged from Homo sapiens (61% query coverage and 80% identity). Further, immunoinformatics analysis of hydrophilicity and flexibility confirmed the antigenicity of the peptide fragment. The linear epitope (P1P2) was synthesized (98% purity) and the purity was confirmed by high-performance liquid chromatography and MS. The indirect Enzyme linked immunosorbent assay results confirmed the presence of the corresponding antibody in VL patient's sera but not in those of healthy and other diseases. The result demonstrated a sensitivity 90%; Se Cl95% (82.16-96.27)% and specificity 100%; Sp Cl95% (84.56-100)% which indicated the possibility to be used as a diagnostic tool. Sensitivity, specificity, and diagnostic efficiency of colloidal gold conjugated anti-P1P2 antibody ICT strip was 100, 95.2, and 96.7%, respectively, which is slightly better as compared to other ICT for VL. Though, our result indicated the utility of anti-P1P2 antibody to detect CICs epitopes, a large-scale inspection in endemic and non-endemic area and in different ethnic population is needed for its validation and authentication.

  9. Multi-epitope Models Explain How Pre-existing Antibodies Affect the Generation of Broadly Protective Responses to Influenza

    PubMed Central

    Zarnitsyna, Veronika I.; Lavine, Jennie; Ellebedy, Ali; Ahmed, Rafi; Antia, Rustom

    2016-01-01

    The development of next-generation influenza vaccines that elicit strain-transcendent immunity against both seasonal and pandemic viruses is a key public health goal. Targeting the evolutionarily conserved epitopes on the stem of influenza’s major surface molecule, hemagglutinin, is an appealing prospect, and novel vaccine formulations show promising results in animal model systems. However, studies in humans indicate that natural infection and vaccination result in limited boosting of antibodies to the stem of HA, and the level of stem-specific antibody elicited is insufficient to provide broad strain-transcendent immunity. Here, we use mathematical models of the humoral immune response to explore how pre-existing immunity affects the ability of vaccines to boost antibodies to the head and stem of HA in humans, and, in particular, how it leads to the apparent lack of boosting of broadly cross-reactive antibodies to the stem epitopes. We consider hypotheses where binding of antibody to an epitope: (i) results in more rapid clearance of the antigen; (ii) leads to the formation of antigen-antibody complexes which inhibit B cell activation through Fcγ receptor-mediated mechanism; and (iii) masks the epitope and prevents the stimulation and proliferation of specific B cells. We find that only epitope masking but not the former two mechanisms to be key in recapitulating patterns in data. We discuss the ramifications of our findings for the development of vaccines against both seasonal and pandemic influenza. PMID:27336297

  10. 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.

  11. 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.

  12. Computer aided prediction and identification of potential epitopes in the receptor binding domain (RBD) of spike (S) glycoprotein of MERS-CoV.

    PubMed

    Ali, Mohammad Tuhin; Morshed, Mohammed Monzur; Gazi, Md Amran; Musa, Md Abu; Kibria, Md Golam; Uddin, Md Jashim; Khan, Md Anik Ashfaq; Hasan, Shihab

    2014-01-01

    Middle East Respiratory Syndrome Coronavirus (MERS-CoV) belongs to the coronaviridae family. In spite of several outbreaks in the very recent years, no vaccine against this deadly virus is developed yet. In this study, the receptor binding domain (RBD) of Spike (S) glycoprotein of MERS-CoV was analyzed through Computational Immunology approach to identify the antigenic determinants (epitopes). In order to do so, the sequences of S glycoprotein that belong to different geographical regions were aligned to observe the conservancy of MERS-CoV RBD. The immune parameters of this region were determined using different in silico tools and Immune Epitope Database (IEDB). Molecular docking study was also employed to check the affinity of the potential epitope towards the binding cleft of the specific HLA allele. The N-terminus RBD (S367-S606) of S glycoprotein was found to be conserved among all the available strains of MERS-CoV. Based on the lower IC50 value, a total of eight potential T-cell epitopes and 19 major histocompatibility complex (MHC) class-I alleles were identified for this conserved region. A 9-mer epitope CYSSLILDY displayed interactions with the maximum number of MHC class-I molecules and projected the highest peak in the B-cell antigenicity plot which concludes that it could be a better choice for designing an epitope based peptide vaccine against MERSCoV considering that it must undergo further in vitro and in vivo experiments. Moreover, in molecular docking study, this epitope was found to have a significant binding affinity of -8.5 kcal/mol towards the binding cleft of the HLA-C*12:03 molecule.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Identification of relevant properties for epitopes detection using a regression model.

    PubMed

    Ambroise, Jérôme; Giard, Joachim; Gala, Jean-Luc; Macq, Benoit

    2011-01-01

    A B-cell epitope is a part of an antigen that is recognized by a specific antibody or B-cell receptor. Detecting the immunogenic region of the antigen is useful in numerous immunodetection and immunotherapeutics applications. The aim of this paper is to find relevant properties to discriminate the location of potential epitopes from the rest of the protein surface. The most relevant properties, identified using two evaluation approaches, are the geometric properties, followed by the conservation score and some chemical properties, such as the proportion of glycine. The selected properties are used in a patch-based epitope localization method including a Single-Layer Perceptron for regression. The output of this Single-Layer Perceptron is used to construct a probability map on the antigen surface. The predictive performances of the method are assessed by computing the AUC using cross validation on two benchmark data sets and by computing the AUC and the precision for a third independent test set.

  5. Immunization against a Saccharide Epitope Accelerates Clearance of Experimental Gonococcal Infection

    PubMed Central

    Gulati, Sunita; Zheng, Bo; Reed, George W.; Su, Xiaohong; Cox, Andrew D.; St. Michael, Frank; Stupak, Jacek; Lewis, Lisa A.; Ram, Sanjay; Rice, Peter A.

    2013-01-01

    The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a TH1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with

  6. Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus

    PubMed Central

    Srivastava, Vikram; Yang, Zheng; Hung, Ivan Fan Ngai; Xu, Jianqing; Zheng, Bojian

    2013-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection. PMID:23487456

  7. 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.

  8. 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.

  9. 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

  10. The Identification and Characterization of Two Novel Epitopes on the Nucleocapsid Protein of the Porcine Epidemic Diarrhea Virus

    PubMed Central

    Wang, Kang; Xie, Chun; Zhang, Jianan; Zhang, Wenchao; Yang, Deqiang; Yu, Lingxue; Jiang, Yifeng; Yang, Shen; Gao, Fei; Yang, Zhibiao; Zhou, Yanjun; Tong, Guangzhi

    2016-01-01

    Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death, particularly in neonatal piglets. The nucleocapsid protein (N protein) of PEDV presents strong immunogenicity and contributes to the cross-reactivity between PEDV and TGEV. However, the characterization of epitopes on the PEDV N protein remains largely unknown. Here, two monoclonal antibodies (MAbs) specific to the N protein of a PEDV strain, FJzz1/2011, were generated and screened against a partially overlapping library of 24 GST-fusion N protein-truncated constructs. We confirmed that residues 18–133 (designated NEP-D4) and residues 252–262 (designated NEP-D6) were the epitopes targeted by MAbs PN-D4 and PN-D6, respectively. Sequence analysis revealed that these two epitopes were highly conserved among PEDV strains but were significantly different from other members of the Coronavirinae subfamily. Western blot analysis showed that they could be specifically recognized by PEDV antisera but could not be recognized by TGEV hyperimmune antisera. Indirect immunofluorescence (IFA) assays confirmed no cross-reaction between these two MAbs and TGEV. In addition, the freeze-thaw cycle and protease treatment results indicated that NEP-D4 was intrinsically disordered. All these results suggest that these two novel epitopes and their cognate MAbs could serve as the basis for the development of precise diagnostic assays for PEDV. PMID:27991537

  11. Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

    PubMed Central

    Chen, Weizao; Yuan, Xiaohui; Chong, Huihui; Prabakaran, Ponraj; Dimitrov, Dimiter S.; He, Yuxian

    2013-01-01

    M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds. PMID:23776690

  12. Identifying Candidate Targets of Immune Responses in Zika Virus Based on Homology to Epitopes in Other Flavivirus Species

    PubMed Central

    Xu, Xiaojun; Vaughan, Kerrie; Weiskopf, Daniela; Grifoni, Alba; Diamond, Michael S.; Sette, Alessandro; Peters, Bjoern

    2016-01-01

    Introduction: The current outbreak of Zika virus has resulted in a massive effort to accelerate the development of ZIKV-specific diagnostics and vaccines. These efforts would benefit greatly from the definition of the specific epitope targets of immune responses in ZIKV, but given the relatively recent emergence of ZIKV as a pandemic threat, few such data are available. Methods: We used a large body of epitope data for other Flaviviruses that was available from the IEDB for a comparative analysis against the ZIKV proteome in order to project targets of immune responses in ZIKV. Results: We found a significant level of overlap between known antigenic sites from other Flavivirus proteins with residues on the ZIKV polyprotein. The E and NS1 proteins shared functional antibody epitope sites, whereas regions of T cell reactivity were conserved within NS3 and NS5 for ZIKV.  Discussion: Our epitope based analysis provides guidance for which regions of the ZIKV polyprotein are most likely unique targets of ZIKV-specific antibodies, and which targets in ZIKV are most likely to be cross-reactive with other Flavivirus species. These data may therefore provide insights for the development of antibody- and T cell-based ZIKV-specific diagnostics, therapeutics and prophylaxis. PMID:28018746

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags".

    PubMed

    Percival-Alwyn, Jennifer L; England, Elizabeth; Kemp, Benjamin; Rapley, Laura; Davis, Nicola H E; McCarthy, Grant R; Majithiya, Jayesh B; Corkill, Dominic J; Welsted, Sarah; Minton, Kevin; Cohen, E Suzanne; Robinson, Matthew J; Dobson, Claire; Wilkinson, Trevor C I; Vaughan, Tristan J; Groves, Maria A T; Tigue, Natalie J

    2015-01-01

    Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.

  18. Collections Conservation.

    ERIC Educational Resources Information Center

    DeCandido, Robert

    Collections conservation is an approach to the preservation treatment of books and book-like materials that is conceptualized and organized in terms of large groups of materials. This guide is intended to enable a library to evaluate its current collections conservation activities. The introduction describes collections conservation and gives…

  19. 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

  20. Structural characterisation of sporozoite components for a multistage, multi-epitope, anti-malarial vaccine.

    PubMed

    Patarroyo, Manuel E; Cifuentes, Gladys; Rodríguez, Raúl

    2008-01-01

    A totally effective anti-malarial vaccine must contain epitopes derived from multiple proteins found in different stages of the particular parasite involved in invasion. It must therefore include sporozoite molecules able to induce protective immunity thereby blocking the parasite's access to hepatic cells; thrombospondin-related anonymous protein (TRAP) is one of them. Conserved high activity binding peptides (HABPs) attaching themselves to hepatic cells were used in immunisation studies with the highly malaria-susceptible Aotus monkey. However, they had to be modified to render them immunogenic. The changes induced in lead peptide 3D structure were analysed by correlating such substitutions with the induction of high anti-sporozoite antibody levels in the experimental monkey model. The modification induced structural changes in most modified HABPs, changing them from random-coil or distorted type III beta-turn structures to classical type III or III' beta-turn, thereby allowing a better fit into the MHC-II-peptide-TCR complex since they bound with high affinity to purified HLA-DRbeta1* molecules. These are the first (TRAP) conserved HABPs corresponding to functionally active amino acid sequences in sporozoite invasion and mobility which, when modified, were able to induce very high anti-sporozoite antibody responses, leading to suggesting them as components in the first line of defence of a fully-effective, subunit-based, multi-epitope, multi-stage, synthetic anti-malarial vaccine.

  1. Synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes.

    PubMed

    Stoloff, Gregory A; Caparros-Wanderley, Wilson

    2007-09-01

    Influenza causes yearly epidemics of mild disease and, occasionally, pandemics with millions of fatalities. Currently, no vaccine is effective against all influenza strains. Analysis of influenza sequences from animal and human isolates using CLUSTALW and a novel proprietary epitope prediction algorithm identified six conserved T cell-reactive regions in several proteins. Immunisation of transgenic mice with a preparation of these six regions as chemically synthesised peptides (FLU-v) induced a specific HLA-A*0201-mediated CD8(+) T cell response. This T cell population also reacted against human cells infected with three non-related influenza strains, confirming that the identified regions contain epitopes naturally presented by infected human cells and conserved amongst non-related viruses. Moreover, FLU-v immunisation significantly increased survival of transgenic mice against lethal challenge with influenza. Overall, FLU-v represents a promising influenza vaccine candidate, obviating the need for yearly vaccinations and allowing the stockpiling and initiation of a worldwide vaccination program in advance of a pandemic outbreak.

  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. iVAX: An integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines

    PubMed Central

    Moise, Leonard; Gutierrez, Andres; Kibria, Farzana; Martin, Rebecca; Tassone, Ryan; Liu, Rui; Terry, Frances; Martin, Bill; De Groot, Anne S

    2015-01-01

    Computational vaccine design, also known as computational vaccinology, encompasses epitope mapping, antigen selection and immunogen design using computational tools. The iVAX toolkit is an integrated set of tools that has been in development since 1998 by De Groot and Martin. It comprises a suite of immunoinformatics algorithms for triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, eliminating regulatory T cell epitopes, and optimizing antigens for immunogenicity and protection against disease. iVAX has been applied to vaccine development programs for emerging infectious diseases, cancer antigens and biodefense targets. Several iVAX vaccine design projects have had success in pre-clinical studies in animal models and are progressing toward clinical studies. The toolkit now incorporates a range of immunoinformatics tools for infectious disease and cancer immunotherapy vaccine design. This article will provide a guide to the iVAX approach to computational vaccinology. PMID:26155959

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Production of IFN-γ and IL-4 Against Intact Catalase and Constructed Catalase Epitopes of Helicobacter pylori From T-Cells

    PubMed Central

    Ghasemian Safaei, Hajieh; Faghri, Jamshid; Moghim, Sharareh; Nasr Esfahani, Bahram; Fazeli, Hossein; Makvandi, Manoochehr; Adib, Minoo; Rashidi, Niloufar

    2015-01-01

    Background: Helicobacter pylori infection is highly prevalent in the developing countries. It causes gastritis, peptic ulcer disease, and gastrocarcinoma. Treatment with drugs and antibiotics is problematic due to the following reasons: cost, resistance to antibiotics, prolonged treatment and using multiple drugs. Catalase is highly conserved among the Helicobacter species and is important to the survival of the organism. It is expressed in high amounts and is exposed to the surface of this bacterium; therefore it represents a suitable candidate vaccine antigen. Objectives: A suitable approach in H. pylori vaccinology is the administration of epitope based vaccines. Therefore the responses of T-cells (IFN-γ and IL-4 production) against the catalase of H. pylori were determined. Then the quality of the immune responses against intact catalase and three epitopes of catalase were compared. Materials and Methods: In this study, a composition of three epitopes of the H. pylori catalase was selected based on Propred software. The effect of catalase epitopes on T-cells were assayed and immune responses identified. Results: The results of IFN-γ, IL-4 production against antigens, epitopes, and recombinant catalase by T-cells were compared for better understanding of epitope efficiency. Conclusions: The current research demonstrated that epitope sequence stimulates cellular immune responses effectively. In addition, increased safety and potency as well as a reduction in time and cost were advantages of this method. Authors are going to use this sequence as a suitable vaccine candidate for further research on animal models and humans in future. PMID:26862387

  9. A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

    PubMed

    Tamborrini, Marco; Geib, Nina; Marrero-Nodarse, Aniebrys; Jud, Maja; Hauser, Julia; Aho, Celestine; Lamelas, Araceli; Zuniga, Armando; Pluschke, Gerd; Ghasparian, Arin; Robinson, John A

    2015-10-16

    Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20-30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

  10. A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A

    PubMed Central

    Tamborrini, Marco; Geib, Nina; Marrero-Nodarse, Aniebrys; Jud, Maja; Hauser, Julia; Aho, Celestine; Lamelas, Araceli; Zuniga, Armando; Pluschke, Gerd; Ghasparian, Arin; Robinson, John A.

    2015-01-01

    Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae. PMID:26501327

  11. Major Trypanosoma cruzi antigenic determinant in Chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

    PubMed Central

    Mesri, E A; Levitus, G; Hontebeyrie-Joskowicz, M; Dighiero, G; Van Regenmortel, M H; Levin, M J

    1990-01-01

    A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease. PMID:1696282

  12. A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

    SciTech Connect

    Boonsathorn, Naphatsawan; Panthong, Sumolrat; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Yasugi, Mayo; Ono, Ken-ichiro; and others

    2014-09-26

    Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

  13. 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

  14. 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.

  15. 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.

  16. Self-adjuvanting influenza candidate vaccine presenting epitopes for cell-mediated immunity on a proteinaceous multivalent nanoplatform.

    PubMed

    Szurgot, Inga; Szolajska, Ewa; Laurin, David; Lambrecht, Benedicte; Chaperot, Laurence; Schoehn, Guy; Chroboczek, Jadwiga

    2013-09-13

    We exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering a multivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The delivery platform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remarkable endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the most abundant influenza internal protein with the conserved primary structure. Two different M1 immunodominant epitopes were separately inserted in Dd external positions without destroying the particles' dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system were properly presented by human dendritic cells triggering efficient activation of antigen-specific T cells responses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens. These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulate cellular immune responses.

  17. Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus

    PubMed Central

    Olal, Daniel; Kuehne, Ana I.; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L.; Lee, Jeffrey E.; King, Liam B.; Kawaoka, Yoshihiro; Dye, John M.

    2012-01-01

    Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics. PMID:22171276

  18. Identification of three antigen epitopes on the nucleocapsid protein of the genotype C of bovine parainfluenza virus type 3.

    PubMed

    Ren, Jian-Le; Zhu, Yuan-Mao; Zhou, Yue-Hui; Lv, Chuang; Yan, Hao; Ma, Lei; Shi, Hong-Fei; Xue, Fei

    2015-07-09

    Bovine parainfluenza virus type 3 (BPIV3) is an important respiratory tract pathogen for both young and adult cattle. So far, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis. But fine mapping of epitopes of BPIV3 is scant and the antigenic variations among the three genotypes of BPIV3 have not been reported. Nucleocapsid protein (NP) is the most abundant protein in the virion and highly conserved in BPIV3, which is crucial for the induction of protective immunity in host. To identify antigenic determinants of BPIV3 NP, a panel of monoclonal antibodies (mAbs) was tested against a series of overlapping recombinant NP fragments expressed in Escherichia coli. Firstly, six monoclonal antibodies (mAbs) against NP of the genotype C of BPIV3 (BPIV3c) were generated by using the purified BPIV3c strain SD0835 as immunogen and the recombinant NP of SD0835 as screening antigen. Then three antigen epitopes were identified with the six mAbs. One epitope (91)GNNADVKYVIYM(102) was recognized by mAb 5E5. The mAbs 7G5, 7G8, 7G9, and 7H5 were reactive with another epitope (407)FYKPTGG(413). The third epitope (428)ESRGDQDQ(435) was reactive with mAb 6F8. Further analysis showed that the epitope (91-102 amino acids [aa]) was the most conserved and reactive with mAb 5E5 for all three genotypes of BPIV3 and HPIV3. The epitope (407-413 aa) was relatively conserved and reactive with mAbs 7G5, 7G8, 7G9, and 7H5 for BPIV3a, BPIV3c and HPIV3, but not reactive with BPIV3b. The epitope (428-435 aa) was less conserved and was reactive only with mAb 6F8 for BPIV3a and BPIV3c. These results suggested that there were evident antigenic variations among the three genotypes of BPIV3 and HPIV3. The mAb 6F8 could be used to detect BPIV3a and BPIV3c. The mAbs 7G5, 7G8, 7G9, and 7H5 might be used for differentiate BPIV3a, BPIV3c and HPIV3 from BPIV3b. The mAb 5E5 might be used for detecting all three types of BPIV3 and HPIV3. The results in this

  19. MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.

    PubMed

    Comber, Joseph D; Philip, Ramila

    2014-05-01

    Major histocompatibility complex class I (MHC-I) presented peptide epitopes provide a 'window' into the changes occurring in a cell. Conventionally, these peptides are generated by proteolysis of endogenously synthesized proteins in the cytosol, loaded onto MHC-I molecules, and presented on the cell surface for surveillance by CD8(+) T cells. MHC-I restricted processing and presentation alerts the immune system to any infectious or tumorigenic processes unfolding intracellularly and provides potential targets for a cytotoxic T cell response. Therefore, therapeutic vaccines based on MHC-I presented peptide epitopes could, theoretically, induce CD8(+) T cell responses that have tangible clinical impacts on tumor eradication and patient survival. Three major methods have been used to identify MHC-I restricted epitopes for inclusion in peptide-based vaccines for cancer: genetic, motif prediction and, more recently, immunoproteomic analysis. Although the first two methods are capable of identifying T cell stimulatory epitopes, these have significant disadvantages and may not accurately represent epitopes presented by a tumor cell. In contrast, immunoproteomic methods can overcome these disadvantages and identify naturally processed and presented tumor associated epitopes that induce more clinically relevant tumor specific cytotoxic T cell responses. In this review, we discuss the importance of using the naturally presented MHC-I peptide repertoire in formulating peptide vaccines, the recent application of peptide-based vaccines in a variety of cancers, and highlight the pros and cons of the current state of peptide vaccines.

  20. 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.

  1. 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

  2. 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.

  3. Conservation Laws

    NASA Astrophysics Data System (ADS)

    Dewitt, Bryce; Christensen, Steven M.

    In the case of the free particle, we interpreted various components of the energy-momentum-stress density as fluxes of energy and momentum. This interpretation can obviously be extended also to particle ensembles and gases. When we speak of fluxes we usually think of quantities that are conserved. In special relativity, energy and momentum are conserved. In general relativity, they are no longer generally conserved, at least if we do not include the energy and momentum of the gravitational field itself. Nevertheless, their densities and fluxes satisfy a covariant generalization of a true conservation law, which is quite easy to obtain.

  4. 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.

  5. 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.

  6. 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

  7. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    PubMed

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Li, Jianfan; Li, Yanhan; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Conservation Tillage

    NASA Astrophysics Data System (ADS)

    Gebhardt, Maurice R.; Daniel, Tommy C.; Schweizer, Edward E.; Allmaras, Raymond R.

    1985-11-01

    Conservation production systems combine tillage and planting practices to reduce soil erosion and loss of water from farmland. Successful conservation tillage practices depend on the ability of farm managers to integrate sound crop production practices with effective pest management systems. More scientific information is needed to determine the relations between tillage practices and physical, chemical, and biological soil factors that affect plant and pest ecology. There is a need to devise improved pest management strategies for conservation tillage and to better understand the impact of conservation tillage on water quality, especially as it is related to use of agricultural chemicals. While savings in fuel, labor, and soil have induced many farmers to adopt conservation tillage, improved methods and equipment should increase adoption even more.

  15. Immunogenicity of multi-epitope-based vaccine candidates administered with the adjuvant Gp96 against rabies.

    PubMed

    Niu, Yange; Liu, Ye; Yang, Limin; Qu, Hongren; Zhao, Jingyi; Hu, Rongliang; Li, Jing; Liu, Wenjun

    2016-04-01

    Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canine-Gp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5-6 IU/mL in mice and 7-9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%-80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.

  16. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex. PMID:21729328

  17. Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages

    PubMed Central

    Machkovech, Heather M.; Bedford, Trevor; Suchard, Marc A.

    2015-01-01

    ABSTRACT Numerous experimental studies have demonstrated that CD8+ T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8+ T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal

  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. 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.

  20. 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.

  1. 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

  2. Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

    PubMed

    Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

    2013-04-01

    Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections.

  3. 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

  4. 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.

  5. Energy Conservation

    ERIC Educational Resources Information Center

    Abelson, Philip H.

    1972-01-01

    Comments on The Potential for Energy Conservation,'' a study by the Office of Emergency Preparedness, emphasizing the coming dependence on foreign oil, and presses for government influence to encourage development of more efficient cars. (AL)

  6. Conservation Presentation.

    ERIC Educational Resources Information Center

    Friday, Gerald

    2001-01-01

    Introduces a project in which students teach about the importance of recycling and conservation by presenting demonstrations. Includes demonstrations on water, plastic, and other recycling products such as steel. (YDS)

  7. Prediction and In Silico Identification of Novel B-Cells and T-Cells Epitopes in the S1-Spike Glycoprotein of M41 and CR88 (793/B) Infectious Bronchitis Virus Serotypes for Application in Peptide Vaccines

    PubMed Central

    Hair Bejo, Mohd; Kadkhodaei, Saeid

    2016-01-01

    Bioinformatic analysis was used to predict antigenic B-cell and T-cell epitopes within the S1 glycoprotein of M41 and CR88 IBV strains. A conserved linear B-cell epitope peptide, YTSNETTDVTS175–185, was identified in M41 IBV strains while three such epitopes types namely, VSNASPNSGGVD279–290, HPKCNFRPENI328–338, and NETNNAGSVSDCTAGT54–69, were predicted in CR88 IBV strains. Analysis of MHCI binding peptides in M41 IBV strains revealed the presence of 15 antigenic peptides out of which 12 were highly conserved in 96–100% of the total M41 strains analysed. Interestingly three of these peptides, GGPITYKVM208, WFNSLSVSI356, and YLADAGLAI472, relatively had high antigenicity index (>1.0). On the other hand, 11 MHCI binding epitope peptides were identified in CR88 IBV strains. Of these, five peptides were found to be highly conserved with a range between 90% and 97%. However, WFNSLSVSL358, SYNISAASV88, and YNISAASVA89 peptides comparably showed high antigenicity scores (>1.0). Combination of antigenic B-cells and T-cells peptides that are conserved across many strains as approach to evoke humoral and CTL immune response will potentially lead to a broad-based vaccine that could reduce the challenges in using live attenuated vaccine technology in the control of IBV infection in poultry. PMID:27667997

  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. 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

  10. CD8 and CD4 epitope predictions in RV144: no strong evidence of a T-cell driven sieve effect in HIV-1 breakthrough sequences from trial participants.

    PubMed

    Dommaraju, Kalpana; Kijak, Gustavo; Carlson, Jonathan M; Larsen, Brendan B; Tovanabutra, Sodsai; Geraghty, Dan E; Deng, Wenjie; Maust, Brandon S; Edlefsen, Paul T; Sanders-Buell, Eric; Ratto-Kim, Silvia; deSouza, Mark S; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttihum, Punnee; Kaewkungwal, Jaranit; O'Connell, Robert J; Robb, Merlin L; Michael, Nelson L; Mullins, James I; Kim, Jerome H; Rolland, Morgane

    2014-01-01

    The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84-91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.

  11. CD8 and CD4 Epitope Predictions in RV144: No Strong Evidence of a T-Cell Driven Sieve Effect in HIV-1 Breakthrough Sequences from Trial Participants

    PubMed Central

    Dommaraju, Kalpana; Kijak, Gustavo; Carlson, Jonathan M.; Larsen, Brendan B.; Tovanabutra, Sodsai; Geraghty, Dan E.; Deng, Wenjie; Maust, Brandon S.; Edlefsen, Paul T.; Sanders-Buell, Eric; Ratto-Kim, Silvia; deSouza, Mark S.; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttihum, Punnee; Kaewkungwal, Jaranit; O'Connell, Robert J.; Robb, Merlin L.; Michael, Nelson L.; Mullins, James I.; Kim, Jerome H.; Rolland, Morgane

    2014-01-01

    The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84–91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants. PMID:25350851

  12. Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

    PubMed Central

    Fu, Fang; Jiang, Fucheng; Wang, Xiangling; Zhang, Xueyun; Wang, Zhuo; Li, Xi

    2014-01-01

    Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched 469KTPAEAR475 of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis. PMID:24416241

  13. Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers

    PubMed Central

    Wan, Hongquan; Yang, Hua; Shore, David A.; Garten, Rebecca J.; Couzens, Laura; Gao, Jin; Jiang, Lianlian; Carney, Paul J.; Villanueva, Julie; Stevens, James; Eichelberger, Maryna C.

    2015-01-01

    A(H1N1)pdm09 influenza A viruses predominated in the 2013–2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses. PMID:25668439

  14. Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment.

    PubMed

    Perschinka, Hannes; Wellenzohn, Bernd; Parson, Walther; van der Zee, Ruurd; Willeit, Johann; Kiechl, Stefan; Wick, Georg

    2007-09-01

    Autoimmune reactions to HSP60 are believed to play a key role during development of early atherosclerosis. Due to the high degree of phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for inducing cross-reactivity to self HSP60, which is expressed on the surface of arterial endothelial cells stressed by classical atherosclerosis risk factors. Conformational epitopes recognized by polyclonal anti-mycobacterial HSP60 antibodies from subjects with atherosclerosis were identified using a phage displayed random library of cyclic constrained 7mer peptides. After five rounds of selection, DNA sequencing of strongly binding clones revealed that one peptide motif (CIGSPSTNC) was present in 64% of all clones, and a second motif (CSFHYQNRC) in 14%. Using a newly developed method for structural alignment of small constrained peptides onto a protein surface, we located the motif present in 14% of all clones on the surface of mycobacterial HSP60. The motif present in 64% of all clones was found on the surface of mycobacterial HSP60 as well as in the homologous region of human HSP60, which makes this epitope a promising candidate for further investigations on cross-reactive epitopes involved in early atherogenesis.

  15. 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.

  16. 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

  17. 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.

  18. Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection.

    PubMed

    Rodrigo, Chaturaka; Walker, Melanie R; Leung, Preston; Eltahla, Auda A; Grebely, Jason; Dore, Gregory J; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D; Cox, Andrea L; Osburn, William; Kim, Arthur Y; Schinkel, Janke; Shoukry, Naglaa H; Lauer, Georg M; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R; Bull, Rowena A

    2017-04-01

    Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (<180days) and chronic (>180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

  19. Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

    PubMed Central

    Han, Thomas; Sui, Jianhua; Bennett, Andrew S.; Liddington, Robert C.; Donis, Ruben O.; Zhu, Quan; Marasco, Wayne A.

    2011-01-01

    Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines. PMID:21569761

  20. Epitope mapping of monoclonal antibody HPT-101: a study combining dynamic force spectroscopy, ELISA and molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Stangner, Tim; Angioletti-Uberti, Stefano; Knappe, Daniel; Singer, David; Wagner, Carolin; Hoffmann, Ralf; Kremer, Friedrich

    2015-12-01

    By combining enzyme-linked immunosorbent assay (ELISA) and optical tweezers-assisted dynamic force spectroscopy (DFS), we identify for the first time the binding epitope of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to the Alzheimer's disease relevant peptide tau[pThr231/pSer235] on the level of single amino acids. In particular, seven tau isoforms are synthesized by replacing binding relevant amino acids by a neutral alanine (alanine scanning). From the binding between mAb HPT-101 and the alanine-scan derivatives, we extract specific binding parameters such as bond lifetime {τ }0, binding length {x}{ts}, free energy of activation {{Δ }}G (DFS) and affinity constant {K}{{a}} (ELISA, DFS). Based on these quantities, we propose criteria to identify essential, secondary and non-essential amino acids, being representative of the antibody binding epitope. The obtained results are found to be in full accord for both experimental techniques. In order to elucidate the microscopic origin of the change in binding parameters, we perform molecular dynamics (MD) simulations of the free epitope in solution for both its parent and modified form. By taking the end-to-end distance {d}{{E}-{{E}}} and the distance between the α-carbons {d}{{C}-{{C}}} of the phosphorylated residues as gauging parameters, we measure how the structure of the epitope depends on the type of substitution. In particular, whereas {d}{{C}-{{C}}} is sometimes conserved between the parent and modified form, {d}{{E}-{{E}}} strongly changes depending on the type of substitution, correlating well with the experimental data. These results are highly significant, offering a detailed microscopic picture of molecular recognition.

  1. 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.

  2. 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.

  3. 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.

  4. Inability to induce consistent T-cell responses recognizing conserved regions within HIIV-1 antigens: a potential mechanism for lack of vaccine efficacy in the step study

    SciTech Connect

    Korber, Bette; Szinger, James

    2009-01-01

    T cell based vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a high probability of matching the epitope induced by vaccination with the infecting viral strain. We compared the frequency and specificity of the CTL epitopes elicited by the replication defective AdS gag/pol/nef vaccine used in the STEP trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. On average vaccination elicited only one epitope per gene. Importantly, the highly conserved epitopes in gag, pol, and nef (> 80% of strains in the current collection of the Los Alamos database [www.hiv.lanl.gov]) were rarely elicited by vaccination. Moreover there was a statistically significant skewing of the T cell response to relative variable epitopes of each gene; only 20% of persons possessed > 3 T cell responses to epitopes likely to be found in circulating strains in the CladeB populations in which the Step trial was conducted. This inability to elicit T cell responses likely to be found in circulating viral strains is a likely factor in the lack of efficacy of the vaccine utilized in the STEP trial. Modeling of the epitope specific responses elicited by vaccination, we project that a median of 8-10 CD8+ T cell epitopes are required to provide >80% likelihood of eliciting at least 3 CD8+ T cell epitopes that would be found on a circulating population of viruses. Development of vaccine regimens which elicit either a greater breadth of responses or elicit responses to conserved regions of the HIV-1 genome are needed to fully evaluate the concept of whether induction of T cell immunity can alter HIV-1 in vivo.

  5. 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

  6. The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

    PubMed Central

    Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling

    2015-01-01

    ABSTRACT NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. IMPORTANCE The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. [Conservation Units.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

    Each of the six instructional units deals with one aspect of conservation: forests, water, rangeland, minerals (petroleum), and soil. The area of the elementary school curriculum with which each correlates is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the…

  12. [Conservation Units.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

    Instructional units deal with each aspect of conservation: forests, wildlife, rangelands, water, minerals, and soil. The area of the secondary school curriculum with which each is correlated is indicated. Lists of general and specific objectives are followed by suggested teaching procedures, including ideas for introducing the topic, questions to…

  13. Marketing Conservation.

    ERIC Educational Resources Information Center

    Ellis, William B.

    1987-01-01

    In 1986, Northeast Utilities began helping shool administrators combat school building energy wastage through a program called Energy Alliance. The typical school can reduce its energy bill by 30 percent by adopting a wide range of conservation measures, including cogeneration, relamping, and energy audits. (MLH)

  14. Lighting Conservation

    ERIC Educational Resources Information Center

    Arnold, Frank D.

    1975-01-01

    With the energy crisis has come an awareness of wasteful consumption practices. One area where research is being done is in lighting conservation. Information in this article is concerned with finding more effective and efficient lighting designs which include daylight utilization, task-oriented lighting, and lighting controls. (MA)

  15. Colorful Conservation

    ERIC Educational Resources Information Center

    Skophammer, Karen

    2011-01-01

    Some people only think about conservation on Earth Day. Being in the "art business" however, this author is always conscious of the many products she thinks get wasted when they could be reused, recycled, and restored--especially in a school building and art room. In this article, she describes an art lesson that allows students to paint…

  16. 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.

  17. 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

  18. 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.

  19. [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.

  20. 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.

  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. Heron conservation

    USGS Publications Warehouse

    Kushlan, J.A.; Hafner, H.

    2000-01-01

    Herons are large, popular and, in many cases, spectacular birds found in wetlands world-wide, both tropical and temperate, natural and man-made. Some populations are very small and localized, some have decreased, some have expanded their ranges, and a few are pests of human activities. In the fifteen years since the publication of the latest monographic treatment of the family, The Herons Handbook, there has been a tremendous increase in our knowledge of heron status and conservation requirements, set against a backdrop of increasing concern about the future of the world?s wetland habitats. This book provides a comprehensive update following two distinct threads. The status and conservation needs of herons are first presented on a regional basis, in a series of chapters set at a continental or subcontinental scale. Over 200 biologists and heron conservationists have contributed to the data summarized here, and the very latest census and survey results provide the most up-to-date and detailed picture of heron populations currently available. Chapters discussing several critical issues in heron conservation follow, tending to focus on the international nature of the problems.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Identification and characterization of CD4⁺ T-cell epitopes on GapC protein of Streptococcus dysgalactiae.

    PubMed

    Yao, Di; Zhang, Hua; Wang, Xintong; Yu, Simiao; Wei, Yuhua; Liu, Wei; Wang, Jiannan; Chen, Xiaoting; Zhang, Zhenghai; Sun, Hunan; Yu, Liquan; Ma, Jinzhu; Tong, Chunyu; Song, Baifen; Cui, Yudong

    2016-02-01

    The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4(+) T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4(+) T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4(+) T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4(+) T-cell epitopes, GapC63-77 and GapC96-110, were for the first time identified using CD4(+) T-cells obtained from GapC-immunized BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63-77 and 96-110 induced significant antigen-specific CD4(+) T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4(+) T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63-77 and GapC96-110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4(+) T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

  8. CHARACTERIZATION OF A LIBRARY OF MONOCLONAL ANTIBODIES AGAINST VITELLOGENIN: CONSERVATION OF EPITOPES

    EPA Science Inventory

    The egg yolk precursor, vitellogenin, a complex phospho-lipo-glycoprotein, is the main source of nutrients for the developing embryo of all oviparous animals. Because of this role it has few evolutionary constraints imposed on its structure, varying widely among species. Despite ...

  9. In vivo validation of predicted and conserved T cell epitopes in a swine influenza model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 using inactivated whole-virus, subunit and alpha replicon-based vaccines do not provide broad prot...

  10. 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

  11. Conformational Epitopes Recognized by Protective Anti-Neisserial Surface Protein A Antibodies

    PubMed Central

    Hou, Victor C.; Moe, Gregory R.; Raad, Zyde; Wuorimaa, Tomi; Granoff, Dan M.

    2003-01-01

    NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-stranded β-barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein. PMID:14638771

  12. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    SciTech Connect

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  13. Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

    USGS Publications Warehouse

    Huang, C.; Chien, M.S.; Landolt, M.L.; Batts, W.; Winton, J.

    1996-01-01

    Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

  14. Energy Conservation

    SciTech Connect

    Simpson, P.

    1995-06-01

    There are two fundamental reasons or motivations for energy conservation: (1) economics; and (2) consideration of energy - its sources and availability. Economics speaks for itself and needs little explanation: a project is undertaken, the cost is recovered in a given period of time (we hope) and our company realizes cost savings thereafter. We study and propose a project; we estimate the payback. If approved, we implement the project. Then, we eagerly watch for its effectiveness - for the proposed payback. The second consideration in regard to energy conservation might - in the foreseeable future - become by far the most important - that of availability. Very knowledgeable persons have stated that this - in reality - is the most serious problem facing our nation today. Readily available, reasonably priced energy has given to the US the high form of living experienced today. An interruption in this flow could catapult our nation in an awesome catastrophe. The energy shortage of the late 70`s might be a forerunner of such an experience.

  15. msa-1 and msa-2c gene analysis and common epitopes assessment in Mexican Babesia bovis isolates.

    PubMed

    Borgonio, Veronica; Mosqueda, Juan; Genis, Alma D; Falcon, Alfonso; Alvarez, J Antonio; Camacho, Minerva; Figueroa, Julio V

    2008-12-01

    Babesia bovis msa-1 and msa-2c genes belong to the variable merozoite surface antigen gene family. These genes code for antigenic proteins present on the merozoite surface (MSA) and are involved in the parasite invasion to the bovine erythrocyte. Previous studies carried out on MSA-1 have evidenced antigen allelic variation in B. bovis isolates from similar endemic regions, as well as in isolates from different geographic regions of the world (Argentina, Australia, Israel). Studies conducted on MSA-2c, however, have shown that this antigen is widely conserved on isolates from distinct geographic regions. In this study, it was hypothesized that MSA-1 and MSA-2c antigens would contain common epitopes despite the presence of nucleotide sequence differences found in 13 B. bovis isolates and strains collected in geographically distant regions of Mexico. Bioinformatics analysis of the primary structure from DNA fragments derived from PCR amplification, cloning, and sequencing of msa-1 and msa-2c genes from the 13 B. bovis populations revealed that the msa-1 gene product present in the various isolates tested is less conserved among isolates obtained within a similar geographic region in Mexico (51-99.7% sequence identity). Results obtained by immunoblot analysis of B. bovis protein extracts reacted with a monoclonal antibody to MSA-1 42-kDa antigen, conclusively showed cross-reactive common epitopes only in Mexican isolates having high sequence identity (>/=99%, eight isolates). Sequence analysis and multiple alignment of deduced MSA-2c demonstrated a high degree of sequence identity (90-100%) among the Mexican B. bovis isolates and strains. Immunoblot results using a polyclonal antibody to MSA-2c reacted against the protein extracts recognized conserved epitopes in at least nine of the B. bovis isolates. The results obtained in this study agree with those previously reported by other researchers and confirm that, based in sequence identity conservation in Mexican B

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

    PubMed

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

    2015-01-01

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

  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. 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.

  2. 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

  3. 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

  4. 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.

  5. Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

    PubMed

    Dervillez, Xavier; Klaukien, Volker; Dürr, Ralf; Koch, Joachim; Kreutz, Alexandra; Haarmann, Thomas; Stoll, Michaela; Lee, Donghan; Carlomagno, Teresa; Schnierle, Barbara; Möbius, Kalle; Königs, Christoph; Griesinger, Christian; Dietrich, Ursula

    2010-10-01

    During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

  6. [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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    PubMed Central

    De Paolis, Francesca; Beghetto, Elisa; Spadoni, Andrea; Montagnani, Francesca; Felici, Franco; Oggioni, Marco R; Gargano, Nicola

    2007-01-01

    Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery. PMID:18088426

  16. Identification of protective linear B-cell epitopes on the subolesin/akirin orthologues of Ornithodoros spp. soft ticks.

    PubMed

    Manzano-Román, Raúl; Díaz-Martín, Verónica; Oleaga, Ana; Pérez-Sánchez, Ricardo

    2015-02-18

    Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5-24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. [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.

  2. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

    SciTech Connect

    Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang Shixia; Lu Shan

    2009-09-15

    Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3{sub B}-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

  3. Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection

    PubMed Central

    Kuo, Chih-Feng; Tsao, Nina; Hsieh, I-Chen; Lin, Yee-Shin; Wu, Jiunn-Jong; Hung, Yu-Ting

    2017-01-01

    Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection. PMID:28355251

  4. A six-domain structural model for Escherichia coli translation initiation factor IF2. Characterisation of twelve surface epitopes.

    PubMed

    Mortensen, K K; Kildsgaard, J; Moreno, J M; Steffensen, S A; Egebjerg, J; Sperling-Petersen, H U

    1998-12-01

    The Escherichia coli translation initiation factor IF2 is a 97 kDa protein which interacts with the initiator fMet-tRNAfMet, GTP and the ribosomal subunits during initiation of protein biosynthesis. For structural and functional investigations of the factor, we have raised and characterised monoclonal antibodies against E. coli IF2. Twelve epitopes have been localised at the surface of the protein molecule by three different methods: Interactions of the monoclonal antibodies with nested deletion mutants of IF2, comparison of the relative location of the epitopes in a competition immunoassay and cross-reactivity analyses of the monoclonal antibodies towards IF2 from Salmonella typhimurium, Klebsiella oxytoca, Enterobacter cloacae, Proteus vulgaris, and Bacillus stearothermophilus. These data are combined with predicted secondary structure and discussed in relation to a six-domain structural model for IF2. The model describes IF2 as a slightly elongated molecule with a structurally compact C-terminal domain, a well-conserved central GTP-binding domain, and a highly charged, solvent exposed N-terminal with protruding alpha-helical structures.

  5. Sequence-conserved and antibody-accessible sites in the V1V2 domain of HIV-1 gp120 envelope protein.

    PubMed

    Shmelkov, Evgeny; Grigoryan, Arsen; Krachmarov, Chavdar; Abagyan, Ruben; Cardozo, Timothy

    2014-09-01

    The immune-correlates analysis of the RV144 trial suggested that epitopes targeted by protective antibodies (Abs) reside in the V1V2 domain of gp120. We mapped V1V2 positional sequence variation onto the conserved V1V2 structural fold and showed that while most of the solvent-accessible V1V2 amino acids vary between strains, there are two accessible molecular surface regions that are conserved and also naturally antigenic. These sites may contain epitopes targeted by broadly cross-reactive anti-V1V2 antibodies.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Development of Peptide-Based Lineage-Specific Serology for Chronic Chagas Disease: Geographical and Clinical Distribution of Epitope Recognition

    PubMed Central

    Bhattacharyya, Tapan; Falconar, Andrew K.; Luquetti, Alejandro O.; Costales, Jaime A.; Grijalva, Mario J.; Lewis, Michael D.; Messenger, Louisa A.; Tran, Trang T.; Ramirez, Juan-David; Guhl, Felipe; Carrasco, Hernan J.; Diosque, Patricio; Garcia, Lineth; Litvinov, Sergey V.; Miles, Michael A.

    2014-01-01

    Background Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI–TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual's history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues. Methodology/Principal Findings We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70%) of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001). Among northern chagasic sera 4/20 (20%) from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology. Conclusions

  12. 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.

  13. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

    PubMed

    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  14. Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

    PubMed Central

    de la Paz, P; Sutton, B J; Darsley, M J; Rees, A R

    1986-01-01

    Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2423326

  15. Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding.

    PubMed Central

    Wyatt, R; Moore, J; Accola, M; Desjardin, E; Robinson, J; Sodroski, J

    1995-01-01

    The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A

  16. 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.

  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. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies.

    PubMed

    Rothbart, Scott B; Lin, Shu; Britton, Laura-Mae; Krajewski, Krzysztof; Keogh, Michael-C; Garcia, Benjamin A; Strahl, Brian D

    2012-01-01

    Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

  20. 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.

  1. Broadly neutralizing alphavirus antibodies bind an epitope on E2 and inhibit entry and egress

    PubMed Central

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K.S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    SUMMARY We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O’nyong’nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern. PMID:26553503

  2. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    PubMed

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

  3. Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes.

    PubMed

    Muryoi, T; Kasturi, K N; Kafina, M J; Cram, D S; Harrison, L C; Sasaki, T; Bona, C A

    1992-04-01

    We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.

  4. Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes

    PubMed Central

    1992-01-01

    We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease. PMID:1372644

  5. Two distinct EF-Tu epitopes induce immune responses in rice and Arabidopsis.

    PubMed

    Furukawa, Takehito; Inagaki, Hiroaki; Takai, Ryota; Hirai, Hiroyuki; Che, Fang-Sik

    2014-02-01

    Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI). We previously reported that rice recognizes flagellin from the rice-incompatible N1141 strain of Acidovorax avenae and subsequently induces immune responses. Cell extracts isolated from flagellin-deficient N1141 (Δfla1141) still induced PTI responses, suggesting that Δfla1141 possesses an additional PAMP distinct from flagellin. Here, we show that elongation factor Tu (EF-Tu), one of the most abundant bacterial proteins, acts as a PAMP in rice and causes several PTI responses. In Brassicaceae species, EF-Tu and an N-acetylated peptide comprising the first 18 amino acids of the N-terminus, termed elf18, are fully active as inducers of PTI responses. By contrast, elf18 did not cause any immune responses in rice, whereas an EF-Tu middle region comprising Lys176 to Gly225, termed EFa50, is fully active as a PAMP in rice. In the leaves of rice plants, EF-Tu induced H2O2 generation and callose deposition, and also triggered resistance to coinfection with pathogenic bacteria. Taken together, these data demonstrate that rice recognizes EFa50, which is distinct from elf18, and that this epitope induces PTI responses.

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

    PubMed Central

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

    2013-01-01

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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces.

    PubMed

    Rajagopal, Srivats; Meyer, Scott C; Goldman, Aaron; Zhou, Min; Ghosh, Indraneel

    2006-11-08

    New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV

    PubMed Central

    Hu, Xintao; Valentin, Antonio; Dayton, Frances; Kulkarni, Viraj; Alicea, Candido; Rosati, Margherita; Chowdhury, Bhabadeb; Gautam, Rajeev; Broderick, Kate E.; Sardesai, Niranjan Y.; Martin, Malcolm A.; Mullins, James I.

    2016-01-01

    HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag. Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. PMID:27733554

  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. A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE.

    PubMed Central

    Buratti, E; Tisminetzky, S G; D'Agaro, P; Baralle, F E

    1997-01-01

    We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17. PMID:9032383

  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 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  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. Conservation potential of agricultural water conservation subsidies

    NASA Astrophysics Data System (ADS)

    Huffaker, Ray

    2008-07-01

    A current policy subsidizes farmers to invest in improved on-farm irrigation efficiency, expecting water to be conserved off farm. Contrary to expectation, water has been increasingly depleted in some regions after such improvements. This paper investigates the policy's failure to conserve water consistently by (1) formulating an economic model of irrigated crop production to determine a profit-maximizing irrigator's range of responses to a subsidy and (2) embedding these responses into hypothetical streamflow diagrams to ascertain their potential to conserve water under various hydrologic regimes. Testable hypotheses are developed to predict the conservation potential of a subsidy in real-world application.

  2. 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.

  3. 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.

  4. Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083

    PubMed Central

    Walsh, Stephen R.; Moodie, Zoe; Fiore-Gartland, Andrew J.; Morgan, Cecilia; Wilck, Marissa B.; Hammer, Scott M.; Buchbinder, Susan P.; Kalams, Spyros A.; Goepfert, Paul A.; Mulligan, Mark J.; Keefer, Michael C.; Baden, Lindsey R.; Swann, Edith M.; Grant, Shannon; Ahmed, Hasan; Li, Fusheng; Hertz, Tomer; Self, Steven G.; Friedrich, David; Frahm, Nicole; Liao, Hua-Xin; Montefiori, David C.; Tomaras, Georgia D.; McElrath, M. Juliana; Hural, John; Graham, Barney S.; Jin, Xia

    2016-01-01

    Background. Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. Methods. A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. Results. T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6–1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2–5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). Conclusions. These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. Clinical Trials Registration. NCT01095224. PMID:26475930

  5. 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

  6. A Novel Universal Neutralizing Monoclonal Antibody against Enterovirus 71 That Targets the Highly Conserved “Knob” Region of VP3 Protein

    PubMed Central

    Meng, Tao; Chow, Vincent Tak Kwong; Kwang, Jimmy

    2014-01-01

    Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the “knob” region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71. PMID:24875055

  7. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine

    PubMed Central

    Fu, Yuanfang; Li, Pinghua; Cao, Yimei; Wang, Na; Sun, Pu; Shi, Qian; Ji, Xincheng; Bao, Huifang; Li, Dong; Chen, Yingli; Bai, Xingwen; Ma, Xueqing; Zhang, Jing; Lu, Zengjun; Liu, Zaixin

    2017-01-01

    Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved “AEKNPLE” epitope spanning amino acids 109–115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant “AEKNPLE” epitope in NSP 3A of FMDV. PMID:28107470

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

    SciTech Connect

    Pan, Ruimin; Chen, Yuxin; Vaine, Michael; Hu, Guangnan; Wang, Shixia; Lu, Shan; Kong, Xiang -Peng

    2015-07-15

    The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitope peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Identification and characterization of a monoclonal antibody recognizing the linear epitope RVADVI on VP1 protein of enterovirus 71.

    PubMed

    Man-Li, Tang; Szyporta, Milene; Fang, Lim Xiao; Kwang, Jimmy

    2012-10-01

    Several large outbreaks of hand-foot-mouth disease (HFMD) have occurred in the Asian-Pacific region since 1997, with Enterovirus 71 (EV71) and/or Coxsackievirus A16 (CAV16) as the main causative agents. Despite the close genetic relationship between the two viruses, only EV71 is associated with severe clinical manifestations and deaths. Effective antiviral treatment and vaccines are not available. High-quality monoclonal antibodies (mAbs) are necessary to improve the accuracy of the diagnosis of EV71. In this study, a mAb (designated 1D9) was generated using EV71 C5 strain virus particles as immunogens. Examined by indirect immunofluorescence assay (IFA) and Western blotting, 1D9 detected successfully all 11 subgenotypes of EV71 and showed no cross-reactivity to the four selected subgenogroups of Coxsackieviruses CAV4, CAV6, CAV10, and CAV16. A linear motif, R(3) VADVI(8), which is located at the N-terminus of the EV71 VP1 protein, was identified as the minimal binding region of 1D9. Alignment and comparison of the 1D9-defined epitope sequence against the listed sequences in the NCBI EV71 database indicated that this epitope R(3) VADVI(8) was highly conserved among EV71 strains, while no significant similarity was observed when blasted against the Coxsackieviruses. This suggests that the mAb 1D9 may be useful for the development of a cost-effective and accurate method for surveillance and early differentiation of EV71 from CAV16 infection.

  14. From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK.

    PubMed

    Gourlay, Louise J; Thomas, Rachael J; Peri, Claudio; Conchillo-Solé, Oscar; Ferrer-Navarro, Mario; Nithichanon, Arnone; Vila, Jordi; Daura, Xavier; Lertmemongkolchai, Ganjana; Titball, Richard; Colombo, Giorgio; Bolognesi, Martino

    2015-04-01

    Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small β-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.

  15. Fine Epitope Mapping of the Central Immunodominant Region of Nucleoprotein from Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

    PubMed Central

    Liu, Dongliang; Li, Yang; Zhao, Jing; Deng, Fei; Duan, Xiaomei; Kou, Chun; Wu, Ting; Li, Yijie; Wang, Yongxing; Ma, Ji; Yang, Jianhua; Hu, Zhihong; Zhang, Fuchun; Zhang, Yujiang; Sun, Surong

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, 247FDEAKK252; E2a, 254VEAL257; E2b, 258NGYLNKH264; E3, 267EVDKA271; and E4, 274DSMITN279) identified through the use of rabbit antiserum, and one BCE (E5, 258NGYL261) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV. PMID:25365026

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Conservation: Threatened by Luxury.

    PubMed

    Webb, Thomas J

    2016-06-20

    When animals are traded in lucrative international luxury markets, individuals really do matter to conservation. Identifying the intrinsic and extrinsic factors that make some species especially vulnerable to this kind of threat helps set guidelines for more effective conservation.

  6. Meeting global conservation challenges

    NASA Astrophysics Data System (ADS)

    2016-10-01

    Hot on the heels of last year's Sustainable Development Goals and the Paris Agreement, representatives from the global conservation community met to set the conservation agenda that will help to implement these targets.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Building robust conservation plans.

    PubMed

    Visconti, Piero; Joppa, Lucas

    2015-04-01

    Systematic conservation planning optimizes trade-offs between biodiversity conservation and human activities by accounting for socioeconomic costs while aiming to achieve prescribed conservation objectives. However, the most cost-efficient conservation plan can be very dissimilar to any other plan achieving the set of conservation objectives. This is problematic under conditions of implementation uncertainty (e.g., if all or part of the plan becomes unattainable). We determined through simulations of parallel implementation of conservation plans and habitat loss the conditions under which optimal plans have limited chances of implementation and where implementation attempts would fail to meet objectives. We then devised a new, flexible method for identifying conservation priorities and scheduling conservation actions. This method entails generating a number of alternative plans, calculating the similarity in site composition among all plans, and selecting the plan with the highest density of neighboring plans in similarity space. We compared our method with the classic method that maximizes cost efficiency with synthetic and real data sets. When implementation was uncertain--a common reality--our method provided higher likelihood of achieving conservation targets. We found that χ, a measure of the shortfall in objectives achieved by a conservation plan if the plan could not be implemented entirely, was the main factor determining the relative performance of a flexibility enhanced approach to conservation prioritization. Our findings should help planning authorities prioritize conservation efforts in the face of uncertainty about future condition and availability of sites.

  16. Conservation in Physics.

    ERIC Educational Resources Information Center

    Finkel, Edward

    1991-01-01

    Discussed is the physical concept of conservation as it is framed within the laws of conservation of mass, of momentum, and of energy. The derivation of Ohm's Law as a generalization of the relationship between the observed measurements of voltage and current serves as the exemplar of how conservation theories are formed. (JJK)

  17. Conservation Action Handbook.

    ERIC Educational Resources Information Center

    National Rifle Association, Washington, DC.

    Conservation problems are identified, with some suggestions for action. General areas covered are: Wildlife Conservation, Soil Conservation, Clean Water, Air Pollution Action, and Outdoor Recreation Action. Appendices list private organizations or agencies concerned with natural resource use and/or management, congressional committees considering…

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Exactly conservative integrators

    SciTech Connect

    Shadwick, B.A.; Bowman, J.C.; Morrison, P.J.

    1995-07-19

    Traditional numerical discretizations of conservative systems generically yield an artificial secular drift of any nonlinear invariants. In this work we present an explicit nontraditional algorithm that exactly conserves invariants. We illustrate the general method by applying it to the Three-Wave truncation of the Euler equations, the Volterra-Lotka predator-prey model, and the Kepler problem. We discuss our method in the context of symplectic (phase space conserving) integration methods as well as nonsymplectic conservative methods. We comment on the application of our method to general conservative systems.

  6. Conservation: Toward firmer ground

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The following aspects of energy conservation were reviewed in order to place the problems in proper perspective: history and goals, conservation accounting-criteria, and a method to overcome obstacles. The effect of changing prices and available supplies of energy sources and their causes on consumption levels during the last few decades were described. Some examples of attainable conservation goals were listed and justified. A number of specific criteria applicable to conservation accounting were given. Finally, a discussion was presented to relate together the following aspects of energy conservation: widespread impact, involvement of government, industry, politics, moral and ethical aspects, urgency and time element.

  7. Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity.

    PubMed

    DiPiazza, Anthony; Richards, Katherine; Poulton, Nicholas; Sant, Andrea J

    2017-03-01

    Avian influenza viruses remain a significant concern due to their pandemic potential. Vaccine trials have suggested that humans respond poorly to avian influenza vaccines relative to seasonal vaccines. It is important to understand, first, if there is a general deficiency in the ability of avian hemagglutinin (HA) proteins to generate immune responses and, if so, what underlies this defect. This question is of particular interest because it has been suggested that in humans, the poor immunogenicity of H7 vaccines may be due to a paucity of CD4 T cell epitopes. Because of the generally high levels of cross-reactive CD4 T cells in humans, it is not possible to compare the inherent immunogenicities of avian and seasonal HA proteins in an unbiased manner. Here, we empirically examine the epitope diversity and abundance of CD4 T cells elicited by seasonal and avian HA proteins. HLA-DR1 and HLA-DR4 transgenic mice were vaccinated with purified HA proteins, and CD4 T cells to specific epitopes were identified and quantified. These studies revealed that the diversity and abundance of CD4 T cells specific for HA do not segregate on the basis of whether the HA was derived from human seasonal or avian influenza viruses. Therefore, we conclude that failure in responses to avian vaccines in humans is likely due to a lack of cross-reactive CD4 T cell memory perhaps coupled with competition with or suppression of naive, HA-specific CD4 T cells by memory CD4 T cells specific for more highly conserved proteins.

  8. 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

  9. 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.

  10. Fixism and conservation science.

    PubMed

    Robert, Alexandre; Fontaine, Colin; Veron, Simon; Monnet, Anne-Christine; Legrand, Marine; Clavel, Joanne; Chantepie, Stéphane; Couvet, Denis; Ducarme, Frédéric; Fontaine, Benoît; Jiguet, Frédéric; le Viol, Isabelle; Rolland, Jonathan; Sarrazin, François; Teplitsky, Céline; Mouchet, Maud

    2016-12-10

    The field of biodiversity conservation has recently been criticized as relying on a fixist view of the living world in which existing species constitute at the same time targets of conservation efforts and static states of reference, which is in apparent disagreement with evolutionary dynamics. We reviewed the prominent role of species as conservation units and the common benchmark approach to conservation that aims to use past biodiversity as a reference to conserve current biodiversity. We found that the species approach is justified by the discrepancy between the time scales of macroevolution and human influence and that biodiversity benchmarks are based on reference processes rather than fixed reference states. Overall, we argue that the ethical and theoretical frameworks underlying conservation research are based on macroevolutionary processes, such as extinction dynamics. Current species, phylogenetic, community, and functional conservation approaches constitute short-term responses to short-term human effects on these reference processes, and these approaches are consistent with evolutionary principles.

  11. 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.

  12. 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

  13. 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.

  14. A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

    PubMed Central

    Fuchs, Julian E.; Ackerbauer, Daniela; Moraes, Adolfo H.; Almeida, Fabio C. L.; Lengger, Nina; Hafner, Christine; Ebner, Christof; Radauer, Christian; Liedl, Klaus R.; Valente, Ana Paula; Breiteneder, Heimo

    2015-01-01

    Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. PMID:26579717

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. Construction and immunogenicity of a recombinant swinepox virus expressing a multi-epitope peptide for porcine reproductive and respiratory syndrome virus

    PubMed Central

    Lin, Huixing; Ma, Zhe; Hou, Xin; Chen, Lei; Fan, Hongjie

    2017-01-01

    To characterize neutralizing mimotopes, phages were selected from a 12-mer phage display library using three anti-porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing monoclonal antibodies: (1) A1; (2) A2; and (3) A7. Of these, A2 and A7 recognize the mimotope, P2, which contains the SRHDHIH motif, which has conserved consensus sequences from amino acid positions 156 to 161 in the N-terminal ectodomain of GP3. The artificial multi-epitope gene, mp2, was designed by combining three repeats of the mimotope P2. The resulting sequence was inserted into the swinepox virus (SPV) genome to construct a recombinant swinepox virus (rSPV-mp2). The rSPV-mp2 was able to stably express the multi-epitope peptide, mP2, in vitro. The rSPV-mp2 immunized pigs exhibited a significantly shorter fever duration compared with the wtSPV treated group (P < 0.05). There was an enhanced humoral and cellular immune response, decreased number of PRRSV genomic copies, and a significant reduction in the gross lung pathology (P < 0.05) was observed following PRRSV infection in rSPV-mp2-immunized animals. The results suggest that the recombinant rSPV-mp2 provided pigs with significant protection against PRRSV infection. PMID:28272485

  1. An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle.

    PubMed

    Gao, Mingchun; Zhang, Runxiang; Li, Meng; Li, Shuang; Cao, Yongsheng; Ma, Bo; Wang, Junwei

    2012-02-01

    To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.

  2. Are cases of mumps in vaccinated patients attributable to mismatches in both vaccine T-cell and B-cell epitopes?: An immunoinformatic analysis.

    PubMed

    Homan, E Jane; Bremel, Robert D

    2014-01-01

    Resurgent mumps outbreaks have raised questions about the current efficacy of mumps vaccines. We have applied immunoinformatics techniques based on principal component analysis to evaluate patterns in predicted B-cell linear epitopes, MHC binding affinity and cathepsin cleavage in the hemagglutinin neuraminidase protein of vaccine strains and wild-type mumps isolates. We have mapped predicted MHC-peptide binding for 37 MHC-I and 28 MHC-II alleles and predicted cleavage by cathepsin B, L and S. By all measures we applied Jeryl-Lynn JL5 major strain is an outlier with immunomic features arising from a small number of amino acid changes that distinguish it from other virus strains. Individuals vaccinated with Jeryl-Lynn who are not exposed to wild-type virus until their protective antibody titer has waned may be unable to recall a protective immune response when exposed to wild-type virus. Dependence on serology to evaluate mumps vaccines may have overemphasized the conservation of one neutralizing antibody epitope, at the expense of monitoring other related changes in the HN protein that could affect recall responses.

  3. Broad-Spectrum Inhibition of HIV-1 by a Monoclonal Antibody Directed against a gp120-Induced Epitope of CD4

    PubMed Central

    Burastero, Samuele E.; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo

    2011-01-01

    To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1. PMID:21818294

  4. Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

    PubMed Central

    Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

    2012-01-01

    The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

  5. Identification of a linear B-cell epitope on non-structural protein 12 of porcine reproductive and respiratory syndrome virus, using a monoclonal antibody.

    PubMed

    Bi, Caihong; Shao, Zengyu; Zhang, Yuanfeng; Hu, Liang; Li, Jiangnan; Huang, Li; Weng, Changjiang

    2017-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. The structure and function of PRRSV nonstructural protein 12 (NSP12) is still unknown. In this study, we produced a monoclonal antibody, named as 1E5, against the NSP12 protein of HP (highly pathogenic) -PRRSV strain HuN4. A series of partially overlapping recombinant NSP12 truncations and synthesized peptides were used to define the epitope recognized by 1E5. We found that (130)KANATSMRFH(139) is the minimal linear epitope and that it is highly conserved among some HP-PRRSV isolates of type 2 PRRSV, but not the classical isolates of type 2 PRRSV or the isolates of type 1 PRRSV. Therefore, 1E5 can be used to establish a valuable tool to distinguish infections with HP-PRRSV isolates of type 2 PRRSV from the classical isolates of type 2 PRRSV and type 1 PRRSV.

  6. 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

  7. 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.

  8. 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.

  9. Conservation: Toward firmer ground

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The following aspects of energy conservation were discussed: conservation history and goals, conservation modes, conservation accounting-criteria, and a method to overcome obstacles. The conservation modes tested fall into one of the following categories: reduced energy consumption, increased efficiency of energy utilization, or substitution of one or more forms of energy for another which is in shorter supply or in some sense thought to be of more value. The conservation accounting criteria include net energy reduction, economic, and technical criteria. A method to overcome obstacles includes (approaches such as: direct personal impact (life style, income, security, aspiration), an element of crisis, large scale involvement of environmental, safety, and health issues, connections to big government, big business, big politics, involvement of known and speculative science and technology, appeal to moral and ethical standards, the transient nature of opportunities to correct the system.

  10. Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites: Naturally Acquired Humoral Immune Response and B-Cell Epitope Mapping in Brazilian Amazon Inhabitants.

    PubMed

    Rodrigues-da-Silva, Rodrigo Nunes; Soares, Isabela Ferreira; Lopez-Camacho, Cesar; Martins da Silva, João Hermínio; Perce-da-Silva, Daiana de Souza; Têva, Antônio; Ramos Franco, Antônia Maria; Pinheiro, Francimeire Gomes; Chaves, Lana Bitencourt; Pratt-Riccio, Lilian Rose; Reyes-Sandoval, Arturo; Banic, Dalma Maria; Lima-Junior, Josué da Costa

    2017-01-01

    The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes (p = 0.003; r = 0.315) and inverse correlation with the time elapsed from the last malaria episode (p = 0.031; r = -0.258). Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This

  11. Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites: Naturally Acquired Humoral Immune Response and B-Cell Epitope Mapping in Brazilian Amazon Inhabitants

    PubMed Central

    Rodrigues-da-Silva, Rodrigo Nunes; Soares, Isabela Ferreira; Lopez-Camacho, Cesar; Martins da Silva, João Hermínio; Perce-da-Silva, Daiana de Souza; Têva, Antônio; Ramos Franco, Antônia Maria; Pinheiro, Francimeire Gomes; Chaves, Lana Bitencourt; Pratt-Riccio, Lilian Rose; Reyes-Sandoval, Arturo; Banic, Dalma Maria; Lima-Junior, Josué da Costa

    2017-01-01

    The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes (p = 0.003; r = 0.315) and inverse correlation with the time elapsed from the last malaria episode (p = 0.031; r = −0.258). Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This

  12. Conservation and behavioral neuroendocrinology.

    PubMed

    Cockrem, J F

    2005-11-01

    The total number of threatened species of vertebrates is likely to be more than 10,000, with approximately one quarter of the world's mammal species, one eighth of the birds and one third of the amphibians threatened with extinction. The rate of loss of animal species and hence of biodiversity is increasing and may become even greater as ecosystems become affected by climate change due to global warming. Behavioral neuroendocrinology, which considers interactions between behavior and neuroendocrine function in animals from all vertebrate taxa, can contribute to animal conservation. Research with laboratory animals can address questions in basic biology relevant to conservation and develop methods for use with threatened animals. Field work with free-living animals considers the basic biology of new species and the use of endocrine tools to assess the susceptibility of species to threats. Non-invasive measurements of hormone concentrations, especially fecal steroids, are extensively used to assess reproductive function and the stress status of animals in captive breeding programs and in the wild. Biodiversity and natural selection both depend on individual variation, and conservation programs often work with animals on an individual basis. The consideration of data from individuals is essential in conservation endocrinology. Direct contributions to conservation programs are challenging as study situations are determined by practical conservation concerns. Indirect contributions such as the provision of scientific input to conservation plans and participation in public education programs offer significant benefits for conservation programs. Directly and indirectly, there are many opportunities for behavioral neuroendocrinologists to contribute to conservation.

  13. 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