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Sample records for less-conserved epitopes present

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

    Science.gov (United States)

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

    2011-06-24

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

  2. Feasibility of Cowpea chlorotic mottle virus-like particles as scaffold for epitope presentations

    NARCIS (Netherlands)

    Hassani-Mehraban, A.; Creutzburg, S.; Heereveld, van L.; Kormelink, R.J.M.

    2015-01-01

    Within the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a

  3. Less Conservative ℋ∞ Fuzzy Control for Discrete-Time Takagi-Sugeno Systems

    Directory of Open Access Journals (Sweden)

    Leonardo Amaral Mozelli

    2011-01-01

    Full Text Available New analysis and control design conditions of discrete-time fuzzy systems are proposed. Using fuzzy Lyapunov's functions and introducing slack variables, less conservative conditions are obtained. The controller guarantees system stabilization and ℋ∞ performance. Numerical tests and a practical experiment in Chua's circuit are presented to show the effectiveness.

  4. Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

    KAUST Repository

    Magnacca, A.; Persiconi, I.; Nurzia, E.; Caristi, S.; Meloni, F.; Barnaba, V.; Paladini, F.; Raimondo, D.; Fiorillo, M. T.; Sorrentino, R.

    2012-01-01

    -presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B

  5. Presentation of an immunodominant immediate-early CD8+ T cell epitope resists human cytomegalovirus immunoevasion.

    Directory of Open Access Journals (Sweden)

    Stefanie Ameres

    Full Text Available Control of human cytomegalovirus (HCMV depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1, that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy.

  6. Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein.

    Science.gov (United States)

    Lorente, Elena; Barriga, Alejandro; García-Arriaza, Juan; Lemonnier, François A; Esteban, Mariano; López, Daniel

    2017-10-01

    The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

  7. Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

    KAUST Repository

    Magnacca, A.

    2012-07-17

    Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

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

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

    2016-01-01

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

  9. Epitope prediction methods

    DEFF Research Database (Denmark)

    Karosiene, Edita

    Analysis. The chapter provides detailed explanations on how to use different methods for T cell epitope discovery research, explaining how input should be given as well as how to interpret the output. In the last chapter, I present the results of a bioinformatics analysis of epitopes from the yellow fever...... peptide-MHC interactions. Furthermore, using yellow fever virus epitopes, we demonstrated the power of the %Rank score when compared with the binding affinity score of MHC prediction methods, suggesting that this score should be considered to be used for selecting potential T cell epitopes. In summary...... immune responses. Therefore, it is of great importance to be able to identify peptides that bind to MHC molecules, in order to understand the nature of immune responses and discover T cell epitopes useful for designing new vaccines and immunotherapies. MHC molecules in humans, referred to as human...

  10. Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

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    Tripathi, Prashant; Moinuddin; Dixit, Kiran; Mir, Abdul Rouf; Habib, Safia; Alam, Khursheed; Ali, Asif

    2014-07-01

    Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

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    Riedl, Petra; Reiser, Michael; Stifter, Katja; Krieger, Jana; Schirmbeck, Reinhold

    2014-07-01

    Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Experimental myositis inducible with transfer of dendritic cells presenting a skeletal muscle C protein-derived CD8 epitope peptide.

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    Okiyama, Naoko; Hasegawa, Hisanori; Oida, Takatoku; Hirata, Shinya; Yokozeki, Hiroo; Fujimoto, Manabu; Miyasaka, Nobuyuki; Kohsaka, Hitoshi

    2015-07-01

    It is suggested that polymyositis, an autoimmune inflammatory myopathy, is mediated by autoaggressive CD8 T cells. Skeletal muscle C protein is a self-antigen that induces C protein-induced myositis, a murine model of polymyositis. To establish a new murine model of myositis inducible with a single CD8 T-cell epitope peptide that derives from the C protein, three internet-based prediction systems were employed to identify 24 candidate peptides of the immunogenic fragment of the C protein and bind theoretically to major histocompatibility complex class I molecules of C57BL/6 (B6) mice. RMA-S cell assay revealed that a HILIYSDV peptide, amino acid position 399-406 of the C protein, had the highest affinity to the H2-K(b) molecules. Transfer of mature bone marrow-derived dendritic cells pulsed with HILIYSDV induced myositis in naive B6 mice. This myositis was suppressed by anti-CD8-depleting antibodies but not by anti-CD4-depleting antibodies. Because this myositis model is mediated by CD8 T cells independently of CD4 T cells, it should be a useful tool to investigate pathology of polymyositis and develop therapies targeting CD8 T cells. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Studies on Nanoparticle Based Avian Influenza Vaccines to Present Immunogenic Epitopes of the Virus with Concentration on Ectodomain of Matrix 2 (M2e) Protein

    Science.gov (United States)

    Babapoor Dighaleh, Sankhiros

    2011-12-01

    Avian influenza is an infectious disease of avian species caused by type A influenza viruses with a significant economic impact on the poultry industry. Vaccination is the main prevention strategy in many countries worldwide. However, available vaccines elicit antibodies against two major surface protein of the virus hemagglutinin (HA) and neuraminidase (NA), where they constantly change by point mutations. Influenza viruses can also easily undergo gene reassortment. Therefore, to protect chickens against new strain of avian influenza virus, as well as control and prevent virus spread among farms, new vaccines needed to be designed which is a tedious, time consuming and expensive. Recently, conserved regions of the influenza genome have been evaluated as possible universal vaccines to eliminate constant vaccine updates based on circulating virus. In this study, peptide nanotechnology was used to generate vaccine nanoparticles that carry the highly conserved external domain of matrix 2 protein (M2e). These nanoparticles presented M2e in monomeric or tetrameric forms, designated as PSC-M2e-CH and BNSC-M2eN-CH. respectively. First, to demonstrate immunogenicity of these nanoparticles, we measured anti-M2e antibody in chickens, particularly when a high dose was applied. Prior to vaccination-challenge study, the challenge dose were determined by oculonasal inoculation of 10 6 EID50 or 107.7 EID50 of low pathogenicity AI virus HSN2 followed by measuring cloacal and tracheal virus shedding. A biphasic virus shedding pattern was observed with two peaks of virus shedding at days 4 and 8 for both tracheal and cloacal swabs. The chickens infected with 107.7 EID50 had significant virus shedding as compared with 106 EID50. Based on results of mentioned studies, a vaccination-challenge study was conducted by using 75mug of each vaccine construct per inoculation (with and without adjuvant) and higher dose of virus for challenge. BN5C-M2e-CH with adjuvant significantly reduced the

  15. Bioinformatics Tools for the Prediction of T-Cell Epitopes

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Nielsen, Morten

    2018-01-01

    T-cell responses are activated by specific peptides, called epitopes, presented on the cell surface by MHC molecules. Binding of peptides to the MHC is the most selective step in T-cell antigen presentation and therefore an essential factor in the selection of potential epitopes. Several in-vitro...

  16. Immune epitope database analysis resource (IEDB-AR)

    DEFF Research Database (Denmark)

    Zhang, Qing; Wang, Peng; Kim, Yohan

    2008-01-01

    We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total...

  17. High epitope expression levels increase competition between T cells.

    Directory of Open Access Journals (Sweden)

    Almut Scherer

    2006-08-01

    Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.

  18. DRREP: deep ridge regressed epitope predictor.

    Science.gov (United States)

    Sher, Gene; Zhi, Degui; Zhang, Shaojie

    2017-10-03

    The ability to predict epitopes plays an enormous role in vaccine development in terms of our ability to zero in on where to do a more thorough in-vivo analysis of the protein in question. Though for the past decade there have been numerous advancements and improvements in epitope prediction, on average the best benchmark prediction accuracies are still only around 60%. New machine learning algorithms have arisen within the domain of deep learning, text mining, and convolutional networks. This paper presents a novel analytically trained and string kernel using deep neural network, which is tailored for continuous epitope prediction, called: Deep Ridge Regressed Epitope Predictor (DRREP). DRREP was tested on long protein sequences from the following datasets: SARS, Pellequer, HIV, AntiJen, and SEQ194. DRREP was compared to numerous state of the art epitope predictors, including the most recently published predictors called LBtope and DMNLBE. Using area under ROC curve (AUC), DRREP achieved a performance improvement over the best performing predictors on SARS (13.7%), HIV (8.9%), Pellequer (1.5%), and SEQ194 (3.1%), with its performance being matched only on the AntiJen dataset, by the LBtope predictor, where both DRREP and LBtope achieved an AUC of 0.702. DRREP is an analytically trained deep neural network, thus capable of learning in a single step through regression. By combining the features of deep learning, string kernels, and convolutional networks, the system is able to perform residue-by-residue prediction of continues epitopes with higher accuracy than the current state of the art predictors.

  19. Prediction of antigenic epitopes on protein surfaces by consensus scoring

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    Zhang Chi

    2009-09-01

    Full Text Available Abstract Background Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance. Results We present a new antigen Epitope Prediction method, which uses ConsEnsus Scoring (EPCES from six different scoring functions - residue epitope propensity, conservation score, side-chain energy score, contact number, surface planarity score, and secondary structure composition. Applied to unbounded antigen structures from an independent test set, EPCES was able to predict antigenic eptitopes with 47.8% sensitivity, 69.5% specificity and an AUC value of 0.632. The performance of the method is statistically similar to other published methods. The AUC value of EPCES is slightly higher compared to the best results of existing algorithms by about 0.034. Conclusion Our work shows consensus scoring of multiple features has a better performance than any single term. The successful prediction is also due to the new score of residue epitope propensity based on atomic solvent accessibility.

  20. Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

    Science.gov (United States)

    Lindesmith, Lisa C; Mallory, Michael L; Debbink, Kari; Donaldson, Eric F; Brewer-Jensen, Paul D; Swann, Excel W; Sheahan, Timothy P; Graham, Rachel L; Beltramello, Martina; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S

    2018-01-01

    Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo -evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle

  1. Immune epitope database analysis resource

    DEFF Research Database (Denmark)

    Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang

    2012-01-01

    The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide......, and the homology mapping tool was updated to enable mapping of discontinuous epitopes onto 3D structures. Furthermore, to serve a wider range of users, the number of ways in which IEDB-AR can be accessed has been expanded. Specifically, the predictive tools can be programmatically accessed using a web interface...

  2. Identification of a novel linear B-cell epitope using a monoclonal antibody against the carboxy terminus of the canine distemper virus nucleoprotein and sequence analysis of the identified epitope in different CDV isolates.

    Science.gov (United States)

    Yi, Li; Cao, Zhigang; Tong, Mingwei; Cheng, Yuening; Yang, Yong; Li, Shuang; Wang, Jianke; Lin, Peng; Sun, Yaru; Zhang, Miao; Cheng, Shipeng

    2017-09-29

    The Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly. In this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401-523). 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 444 GDKYPIHFNDER 455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence. This collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.

  3. Delivery of a MalE CD4+-T-Cell Epitope into the Major Histocompatibility Complex Class II Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase ral NPKSupply

    Czech Academy of Sciences Publication Activity Database

    Loucká, Jiřina; Schlecht, G.; Vojtová, Jana; Leclerc, C.; Šebo, Peter

    2002-01-01

    Roč. 70, č. 2 (2002), s. 1002-1005 ISSN 0019-9567 R&D Projects: GA ČR GA310/01/0934; GA AV ČR IAA5020907; GA MŠk ME 167 Grant - others:QLK2-CT(US) 00556 Institutional research plan: CEZ:AV0Z5020903 Keywords : delivery * epitope * complex Subject RIV: EE - Microbiology, Virology Impact factor: 4.039, year: 2002

  4. Computer-Aided Design of an Epitope-Based Vaccine against Epstein-Barr Virus

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2017-01-01

    Full Text Available Epstein-Barr virus is a very common human virus that infects 90% of human adults. EBV replicates in epithelial and B cells and causes infectious mononucleosis. EBV infection is also linked to various cancers, including Burkitt’s lymphoma and nasopharyngeal carcinomas, and autoimmune diseases such as multiple sclerosis. Currently, there are no effective drugs or vaccines to treat or prevent EBV infection. Herein, we applied a computer-aided strategy to design a prophylactic epitope vaccine ensemble from experimentally defined T and B cell epitopes. Such strategy relies on identifying conserved epitopes in conjunction with predictions of HLA presentation for T cell epitope selection and calculations of accessibility and flexibility for B cell epitope selection. The T cell component includes 14 CD8 T cell epitopes from early antigens and 4 CD4 T cell epitopes, targeted during the course of a natural infection and providing a population protection coverage of over 95% and 81.8%, respectively. The B cell component consists of 3 experimentally defined B cell epitopes from gp350 plus 4 predicted B cell epitopes from other EBV envelope glycoproteins, all mapping in flexible and solvent accessible regions. We discuss the rationale for the formulation and possible deployment of this epitope vaccine ensemble.

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

    Science.gov (United States)

    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.

  6. Immune Epitope Database and Analysis Resource (IEDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — This repository contains antibody/B cell and T cell epitope information and epitope prediction and analysis tools for use by the research community worldwide. Immune...

  7. EpiJen: a server for multistep T cell epitope prediction

    Directory of Open Access Journals (Sweden)

    Guan Pingping

    2006-03-01

    Full Text Available Abstract Background The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP to the endoplasmic reticulum (ER, where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM, EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

  8. Presentations

    International Nuclear Information System (INIS)

    2007-01-01

    The presented materials consist of presentations of international workshop which held in Warsaw from 4 to 5 October 2007. Main subject of the meeting was progress in manufacturing as well as research program development for neutron detector which is planned to be placed at GANIL laboratory and will be used in nuclear spectroscopy research

  9. Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Presentations

    International Nuclear Information System (INIS)

    2007-01-01

    The PARIS meeting held in Cracow, Poland from 14 to 15 May 2007. The main subjects discussed during this meeting were the status of international project dedicated to gamma spectroscopy research. The scientific research program includes investigations of giant dipole resonance, probe of hot nuclei induced in heavy reactions, Jacobi shape transitions, isospin mixing and nuclear multifragmentation. The mentioned programme needs Rand D development such as new scintillations materials as lanthanum chlorides and bromides as well as new photo detection sensors as avalanche photodiodes - such subjects are also subjects of discussion. Additionally results of computerized simulations of scintillation detectors properties by means of GEANT- 4 code are presented

  11. Epitope Mapping of Monoclonal Antibody PMab-38 Against Dog Podoplanin.

    Science.gov (United States)

    Chang, Yao-Wen; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

    2017-12-01

    Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression of PDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediated platelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associated fibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determined using a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38 is Tyr67 and Glu68 of dPDPN.

  12. Presentation

    Directory of Open Access Journals (Sweden)

    Eduardo Vicente

    2013-06-01

    Full Text Available In the present edition of Significação – Scientific Journal for Audiovisual Culture and in the others to follow something new is brought: the presence of thematic dossiers which are to be organized by invited scholars. The appointed subject for the very first one of them was Radio and the invited scholar, Eduardo Vicente, professor at the Graduate Course in Audiovisual and at the Postgraduate Program in Audiovisual Media and Processes of the School of Communication and Arts of the University of São Paulo (ECA-USP. Entitled Radio Beyond Borders the dossier gathers six articles and the intention of reuniting works on the perspectives of usage of such media as much as on the new possibilities of aesthetical experimenting being build up for it, especially considering the new digital technologies and technological convergences. It also intends to present works with original theoretical approach and original reflections able to reset the way we look at what is today already a centennial media. Having broadened the meaning of “beyond borders”, four foreign authors were invited to join the dossier. This is the first time they are being published in this country and so, in all cases, the articles where either written or translated into Portuguese.The dossier begins with “Radio is dead…Long live to the sound”, which is the transcription of a thought provoking lecture given by Armand Balsebre (Autonomous University of Barcelona – one of the most influential authors in the world on the Radio study field. It addresses the challenges such media is to face so that it can become “a new sound media, in the context of a new soundscape or sound-sphere, for the new listeners”. Andrew Dubber (Birmingham City University regarding the challenges posed by a Digital Era argues for a theoretical approach in radio studies which can consider a Media Ecology. The author understands the form and discourse of radio as a negotiation of affordances and

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

    Science.gov (United States)

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

    2017-10-13

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

  14. The Immune Epitope Database 2.0

    DEFF Research Database (Denmark)

    Hoof, Ilka; Vita, R; Zarebski, L

    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...

  15. Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity.

    Science.gov (United States)

    Ichikawa, Kosuke; Kagamu, Hiroshi; Koyama, Kenichi; Miyabayashi, Takao; Koshio, Jun; Miura, Satoru; Watanabe, Satoshi; Yoshizawa, Hirohisa; Narita, Ichiei

    2012-09-21

    MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

  17. The Advantages of Multi-Epitope Tumor Antigens as an Approach to Treating Breast Cancer

    National Research Council Canada - National Science Library

    Kiertscher, Sylvia

    1999-01-01

    .... We hypothesized that the processing and presentation of multiple tumor antigen epitopes by DC is a more efficient and effective way of stimulating T cell responses than current HLA-restricted peptide-based methods...

  18. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

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

    Science.gov (United States)

    Ofran, Yanay; Schlessinger, Avner; Rost, Burkhard

    2008-11-01

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

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

    Science.gov (United States)

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

    2018-01-01

    T-cell epitope structure identification is a significant challenging immunoinformatic problem within epitope-based vaccine design. Epitopes or antigenic peptides are a set of amino acids that bind with the Major Histocompatibility Complex (MHC) molecules. The aim of this process is presented by Antigen Presenting Cells to be inspected by T-cells. MHC-molecule-binding epitopes are responsible for triggering the immune response to antigens. The epitope's three-dimensional (3D) molecular structure (i.e., tertiary structure) reflects its proper function. Therefore, the identification of MHC class-II epitopes structure is a significant step towards epitope-based vaccine design and understanding of the immune system. In this paper, we propose a new technique using a Genetic Algorithm for Predicting the Epitope Structure (GAPES), to predict the structure of MHC class-II epitopes based on their sequence. The proposed Elitist-based genetic algorithm for predicting the epitope's tertiary structure is based on Ab-Initio Empirical Conformational Energy Program for Peptides (ECEPP) Force Field Model. The developed secondary structure prediction technique relies on Ramachandran Plot. We used two alignment algorithms: the ROSS alignment and TM-Score alignment. We applied four different alignment approaches to calculate the similarity scores of the dataset under test. We utilized the support vector machine (SVM) classifier as an evaluation of the prediction performance. The prediction accuracy and the Area Under Receiver Operating Characteristic (ROC) Curve (AUC) were calculated as measures of performance. The calculations are performed on twelve similarity-reduced datasets of the Immune Epitope Data Base (IEDB) and a large dataset of peptide-binding affinities to HLA-DRB1*0101. The results showed that GAPES was reliable and very accurate. We achieved an average prediction accuracy of 93.50% and an average AUC of 0.974 in the IEDB dataset. Also, we achieved an accuracy of 95

  1. 76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

    Science.gov (United States)

    2011-08-18

    ... direct-discovery technology for use in FDA laboratories. C. Eligibility Information The technology...] Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and... technology to molecularly characterize peptide epitopes that are processed and presented on soluble HLA...

  2. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    DEFF Research Database (Denmark)

    Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

    2015-01-01

    of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage...

  3. Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation

    DEFF Research Database (Denmark)

    Willats, William George Tycho; Limberg, G.; Buchholt, H.C.

    2000-01-01

    occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F...

  4. Expitope: a web server for epitope expression.

    Science.gov (United States)

    Haase, Kerstin; Raffegerst, Silke; Schendel, Dolores J; Frishman, Dmitrij

    2015-06-01

    Adoptive T cell therapies based on introduction of new T cell receptors (TCRs) into patient recipient T cells is a promising new treatment for various kinds of cancers. A major challenge, however, is the choice of target antigens. If an engineered TCR can cross-react with self-antigens in healthy tissue, the side-effects can be devastating. We present the first web server for assessing epitope sharing when designing new potential lead targets. We enable the users to find all known proteins containing their peptide of interest. The web server returns not only exact matches, but also approximate ones, allowing a number of mismatches of the users choice. For the identified candidate proteins the expression values in various healthy tissues, representing all vital human organs, are extracted from RNA Sequencing (RNA-Seq) data as well as from some cancer tissues as control. All results are returned to the user sorted by a score, which is calculated using well-established methods and tools for immunological predictions. It depends on the probability that the epitope is created by proteasomal cleavage and its affinities to the transporter associated with antigen processing and the major histocompatibility complex class I alleles. With this framework, we hope to provide a helpful tool to exclude potential cross-reactivity in the early stage of TCR selection for use in design of adoptive T cell immunotherapy. The Expitope web server can be accessed via http://webclu.bio.wzw.tum.de/expitope. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

    Science.gov (United States)

    Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza

    2017-07-01

    Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All

  6. Epitope discovery with phylogenetic hidden Markov models.

    LENUS (Irish Health Repository)

    Lacerda, Miguel

    2010-05-01

    Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation-selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.

  7. Epitope finding in Zika virus molecule:The first world report

    Institute of Scientific and Technical Information of China (English)

    Somsri Wiwanitkit; Viroj Wiwanitkit

    2017-01-01

    Zika virus infection is a new problematic virus infection that becomes the present public health problem. Now this mosquito borne infectious disease can be seen worldwide and can cause dengue-like infection. In addition, it can also induce trans-placental infection and result in congenital neurological defect. To prevent this infec-tion, there is still no specific vaccine. To find a new vaccine, finding epitope is the first step. Here, the authors report the study to find epitope within Zika virus molecule. According to this study, the appropriate epitopes can be seen. This is the first world report on epitope finding for Zika virus. The data can be useful for further vaccine development.

  8. Automatic Generation of Validated Specific Epitope Sets

    Directory of Open Access Journals (Sweden)

    Sebastian Carrasco Pro

    2015-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  10. Microbiota epitope similarity either dampens or enhances the immunogenicity of disease-associated antigenic epitopes.

    Directory of Open Access Journals (Sweden)

    Sebastian Carrasco Pro

    Full Text Available The microbiome influences adaptive immunity and molecular mimicry influences T cell reactivity. Here, we evaluated whether the sequence similarity of various antigens to the microbiota dampens or increases immunogenicity of T cell epitopes. Sets of epitopes and control sequences derived from 38 antigenic categories (infectious pathogens, allergens, autoantigens were retrieved from the Immune Epitope Database (IEDB. Their similarity to microbiome sequences was calculated using the BLOSUM62 matrix. We found that sequence similarity was associated with either dampened (tolerogenic; e.g. most allergens or increased (inflammatory; e.g. Dengue and West Nile viruses likelihood of a peptide being immunogenic as a function of epitope source category. Ten-fold cross-validation and validation using sets of manually curated epitopes and non-epitopes derived from allergens were used to confirm these initial observations. Furthermore, the genus from which the microbiome homologous sequences were derived influenced whether a tolerogenic versus inflammatory modulatory effect was observed, with Fusobacterium most associated with inflammatory influences and Bacteroides most associated with tolerogenic influences. We validated these effects using PBMCs stimulated with various sets of microbiome peptides. "Tolerogenic" microbiome peptides elicited IL-10 production, "inflammatory" peptides elicited mixed IL-10/IFNγ production, while microbiome epitopes homologous to self were completely unreactive for both cytokines. We also tested the sequence similarity of cockroach epitopes to specific microbiome sequences derived from households of cockroach allergic individuals and non-allergic controls. Microbiomes from cockroach allergic households were less likely to contain sequences homologous to previously defined cockroach allergens. These results are compatible with the hypothesis that microbiome sequences may contribute to the tolerization of T cells for allergen

  11. EPMLR: sequence-based linear B-cell epitope prediction method using multiple linear regression.

    Science.gov (United States)

    Lian, Yao; Ge, Meng; Pan, Xian-Ming

    2014-12-19

    B-cell epitopes have been studied extensively due to their immunological applications, such as peptide-based vaccine development, antibody production, and disease diagnosis and therapy. Despite several decades of research, the accurate prediction of linear B-cell epitopes has remained a challenging task. In this work, based on the antigen's primary sequence information, a novel linear B-cell epitope prediction model was developed using the multiple linear regression (MLR). A 10-fold cross-validation test on a large non-redundant dataset was performed to evaluate the performance of our model. To alleviate the problem caused by the noise of negative dataset, 300 experiments utilizing 300 sub-datasets were performed. We achieved overall sensitivity of 81.8%, precision of 64.1% and area under the receiver operating characteristic curve (AUC) of 0.728. We have presented a reliable method for the identification of linear B cell epitope using antigen's primary sequence information. Moreover, a web server EPMLR has been developed for linear B-cell epitope prediction: http://www.bioinfo.tsinghua.edu.cn/epitope/EPMLR/ .

  12. Nanoallergens: A multivalent platform for studying and evaluating potency of allergen epitopes in cellular degranulation

    Science.gov (United States)

    Deak, Peter E; Vrabel, Maura R; Pizzuti, Vincenzo J; Kiziltepe, Tanyel

    2016-01-01

    Degranulation caused by type I hypersensitivity (allergies) is a complex biophysical process, and available experimental models for studying relevant immunoglobulin E binding epitopes on allergen proteins lack the ability to adequately evaluate, rank, and associate these epitopes individually and with each other. In this study, we propose a new allergy model system for studying potential allergen epitopes using nanoallergens, liposomes modified to effectively display IgE binding epitopes/haptens. By utilizing the covalently conjugated lipid tails on two hapten molecules (dinitrophenol and dansyl), hapten molecules were successfully incorporated into liposomes with high precision to form nanoallergens. Nanoallergens, with precisely controlled high-particle valency, can trigger degranulation with much greater sensitivity than commonly used bovine serum albumin conjugates. In rat basophil leukemia cell experiments, nanoallergens with only 2% hapten loading were able to trigger degranulation in vitro at concentrations as low as 10 pM. Additionally, unlike bovine serum albumin-hapten conjugates, nanoallergens allow exact control over particle size and valency. By varying the nanoallergen parameters such as size, valency, monovalent affinity of hapten, and specific IgE ratios, we exposed the importance of these variables on degranulation intensity while demonstrating nanoallergens’ potential for evaluating both high- and low-affinity epitopes. The data presented in this article establish nanoallergen platform as a reliable and versatile allergy model to study and evaluate allergen epitopes in mast cell degranulation. PMID:27188517

  13. Epitope mapping of the domains of human angiotensin converting enzyme.

    Science.gov (United States)

    Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

    2006-06-01

    Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

  14. From Viral genome to specific peptide epitopes - Methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Rasmussen, Michael; Harndahl, Mikkel

    The affinity for and stability of peptides bound by major histocompatibility complex (MHC) class I molecules are instrumental factors in presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). In swine, such peptide presentations by swine leukocyte antigens (SLA) are crucial for swine i...

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

    Science.gov (United States)

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

    2018-07-01

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

  16. Localization of immunodominant linear B-cell epitopes of Vibrio ...

    African Journals Online (AJOL)

    AJL

    2012-05-01

    May 1, 2012 ... In order to locate the epitopes of OmpU protein, epitope prediction was performed ... enzyme linked immunosorbent assay; OmpU, outer membrane protein U .... recombinant plasmids were extracted and identified by PCR,.

  17. Catalase epitopes vaccine design for Helicobacter pylori: A ...

    African Journals Online (AJOL)

    Jane

    2011-08-15

    Aug 15, 2011 ... colored region and P1 anchor or the starting residue of each ..... score of MVNKDVKQTT; * 1, MVNKDVKQTT is a composition of two epitopes: MVNKDVKQT and .... Therapeutic efficacy of a multi-epitope vaccine against.

  18. Plant viruses as scaffolds for the presentation of vaccine epitopes

    Czech Academy of Sciences Publication Activity Database

    Plchová, Helena; Čeřovská, Noemi; Vaculík, Petr; Moravec, Tomáš

    2017-01-01

    Roč. 61, č. 1 (2017), s. 1-12 ISSN 0006-3134 R&D Projects: GA ČR(CZ) GA15-10768S; GA ČR(CZ) GAP501/12/1761 Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : tobacco-mosaic-virus * x coat protein * human-papillomavirus type-16 * green fluorescent protein * n-terminal segment * triple gene block * cell-to-cell * transient expression * nicotiana-benthamiana * viral vector * transient expression * plant viral expression vectors Subject RIV: GE - Plant Breeding OBOR OECD: Virology Impact factor: 1.551, year: 2016

  19. Localization of immunodominant linear B-cell epitopes of Vibrio ...

    African Journals Online (AJOL)

    Outer membrane protein U (OmpU), an adhesion protein of Vibrio mimicus, is a good antigen, but its epitopes are still unclear. In order to locate the epitopes of OmpU protein, epitope prediction was performed using the amino acid sequence of OmpU protein of V. mimicus HX4 strain that was isolated from the diseased ...

  20. Epitope-Specific Tolerance Modes Differentially Specify Susceptibility to Proteolipid Protein-Induced Experimental Autoimmune Encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2017-11-01

    Full Text Available Immunization with myelin components can elicit experimental autoimmune encephalomyelitis (EAE. EAE susceptibility varies between mouse strains, depending on the antigen employed. BL/6 mice are largely resistant to EAE induction with proteolipid protein (PLP, probably a reflection of antigen-specific tolerance. However, the extent and mechanism(s of tolerance to PLP remain unclear. Here, we identified three PLP epitopes in PLP-deficient BL/6 mice. PLP-sufficient mice did not respond against two of these, whereas tolerance was “leaky” for an epitope with weak predicted MHCII binding, and only this epitope was encephalitogenic. In TCR transgenic mice, the “EAE-susceptibility-associated” epitope was “ignored” by specific CD4 T cells, whereas the “resistance-associated” epitope induced clonal deletion and Treg induction in the thymus. Central tolerance was autoimmune regulator dependent and required expression and presentation of PLP by thymic epithelial cells (TECs. TEC-specific ablation of PLP revealed that peripheral tolerance, mediated by dendritic cells through recessive tolerance mechanisms (deletion and anergy, could largely compensate for a lack of central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self.

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

    Science.gov (United States)

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

    2002-10-01

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

  2. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

    Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

    2016-01-01

    Viral envelope glycoproteins are major targets for antibodies that bind to and inactivate viral particles. The capacity of a viral vaccine to induce virus-neutralizing antibodies is often used as a marker for vaccine efficacy. Yet the number of known neutralization target epitopes is restricted o...

  3. In Silico Prediction of T and B Cell Epitopes of Der f 25 in Dermatophagoides farinae

    Directory of Open Access Journals (Sweden)

    Xiaohong Li

    2014-01-01

    Full Text Available The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371. Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224 and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

  4. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.

    Science.gov (United States)

    Hosono-Fukao, Tomomi; Ohtake-Niimi, Shiori; Nishitsuji, Kazuchika; Hossain, Md Motarab; van Kuppevelt, Toin H; Michikawa, Makoto; Uchimura, Kenji

    2011-11-01

    RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and cytokines in central nervous tissues. Chemically modified heparins that lack the trisulfated disaccharides failed to inhibit the RB4CD12 recognition of HS chains. To determine the localization of the RB4CD12 anti-HS epitope in the brain, we performed an immunohistochemical analysis for cryocut sections of mouse brain. The RB4CD12 staining signals were colocalized with laminin and were detected abundantly in the vascular basement membrane. Bacterial heparinases eliminated the RB4CD12 staining signals. The RB4CD12 epitope localization was confirmed by immunoelectron microscopy. Western blotting analysis revealed that the size of a major RB4CD12-positive molecule is ∼460 kDa in a vessel-enriched fraction of the mouse brain. Disaccharide analysis with reversed-phase ion-pair HPLC showed that [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-] trisulfated disaccharide residues are present in HS purified from the vessel-enriched brain fraction. These results indicated that the RB4CD12 anti-HS epitope exists in large quantities in the brain vascular basement membrane. Copyright © 2011 Wiley-Liss, Inc.

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

    Science.gov (United States)

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

    2015-05-01

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

  6. Indirect detection of an epitope-specific response to HIV-1 gp120 immunization in human subjects.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available A specific response of human serum neutralizing antibodies (nAb to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120 of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology--a variation of sieve analysis--compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219.

  7. Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

    Science.gov (United States)

    Rana, Aarti; Thakur, Shweta; Bhardwaj, Nupur; Kumar, Devender; Akhter, Yusuf

    2016-12-01

    For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. High-Throughput Tools for Characterization of Antibody Epitopes

    DEFF Research Database (Denmark)

    Christiansen, Anders

    mapping. In Chapter 1, it was examined whether combining phage display, a traditional epitope mapping approach, with HTS would improve the method. The developed approach was successfully used to map Ara h 1 epitopes in sera from patients with peanut allergy. Notably, the sera represented difficult...... proliferation advantages. Finally, in Chapter 4, a different emerging technology, next-generation peptide microarrays, was applied for epitope mapping of major peanut allergens using sera from allergic patients. New developments in the peptide microarray have enabled a greatly increased throughput....... In this study, these improvements were utilized to characterize epitopes at high resolution, i.e. determine the importance of each residue for antibody binding, for all major peanut allergens. Epitope reactivity among patients often converged on known epitope hotspots, however the binding patterns were somewhat...

  9. Proof of principle for epitope-focused vaccine design

    Science.gov (United States)

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

    2014-03-01

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

  10. Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

    Science.gov (United States)

    Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

    2017-09-01

    It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

  11. Distinct Escape Pathway by Hepatitis C Virus Genotype 1a from a Dominant CD8+ T Cell Response by Selection of Altered Epitope Processing.

    Science.gov (United States)

    Walker, Andreas; Skibbe, Kathrin; Steinmann, Eike; Pfaender, Stephanie; Kuntzen, Thomas; Megger, Dominik A; Groten, Svenja; Sitek, Barbara; Lauer, Georg M; Kim, Arthur Y; Pietschmann, Thomas; Allen, Todd M; Timm, Joerg

    2016-01-01

    Antiviral CD8(+) T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by preexisting viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8(+) T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI1373-1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8(+) T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, the detection of strong CD8(+) T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues upstream of the epitope (S1368P; P = 0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses. HCV is able to evolutionary adapt to CD8(+) T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes, this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids upstream of the

  12. From viral genome to specific peptide epitopes: methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Rasmussen, Michael; Harndah, Mikkel

    2013-01-01

    to predict likely candidates for peptide-SLA binding. These results were combined with binding predictions generated by the algorithm, NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) in order to select peptide candidates for in vitro analysis. The correlation between high affinity and high stability.......000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods...... originally developed for analysis of human leukocyte antigen (HLA) presentation of peptides. The methods presented provide a timely and cost-effective approach to CTL epitope discovery that can be applied to diseases of swine and of other mammalian species of interest....

  13. Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins

    Directory of Open Access Journals (Sweden)

    Wu Shipo

    2012-06-01

    Full Text Available Abstract Background Ebola viruses (EBOVs cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs. Results Computer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV, GPCAGDFAF and LYDRLASTV (Zaire EBOV could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma. Conclusion Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.

  14. Distribution of some pectic and arabinogalactan protein epitopes during Solanum lycopersicum (L.) adventitious root development.

    Science.gov (United States)

    Sala, Katarzyna; Malarz, Katarzyna; Barlow, Peter W; Kurczyńska, Ewa U

    2017-01-25

    The adventitious roots (AR) of plants share the same function as primary and lateral roots (LR), although their development is mainly an adaptive reaction to stress conditions. Regeneration of grafted plants is often accompanied by AR formation thus making the grafting technique a good model for studying AR initiation and development and their means of emergence. Pectins and arabinogalactan proteins (AGP) are helpful markers of particular cellular events, such as programmed cell death (PCD), elongation, proliferation or other differentiation events that accompany AR development. However, little is known about the distribution of pectins and AGPs during AR ontogeny, either in the primordium or stem tissues from which AR arise or their correspondence with these events during LR formation. AR were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment (grafting versus cutting) of the parental tissue. Immunochemical analysis of the presence of pectic (LM8, LM19, LM20) and AGP (JIM8, JIM13, JIM16) epitopes in AR and AR-associated tissues showed differential, tissue-specific distributions of these epitopes. Two pectic epitopes (LM19, LM20) were developmentally regulated and the occurrence of the LM8 xylogalacturonan epitope in the root cap of the AR differed from other species described so far. AGP epitopes were abundantly present in the cytoplasmic compartments (mainly the tonoplast) and were correlated with the degree of cell vacuolisation. JIM8 and JIM13 epitopes were detected in the more advanced stages of primordium development, whereas the JIM16 epitope was present from the earliest division events of the initial AR cells. The comparison between AR and LR showed quantitative (AGP,) and qualitative (pectins) differences. The chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. In AR, developmental changes in the

  15. CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

    Directory of Open Access Journals (Sweden)

    Sylvain Cardinaud

    2011-05-01

    Full Text Available Cytotoxic CD8+ T cells (CTLs play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF. We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D and a majority encoding an asparagine (Q9VF/5N. We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

  16. A xylogalacturonan epitope is specifically associated with plant cell detachment

    DEFF Research Database (Denmark)

    Willats, William George Tycho; McCartney, L.; Steele-King, C.G.

    2004-01-01

    A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitop...

  17. Confirmation of antibodies against L-tryptophan-like epitope in ...

    African Journals Online (AJOL)

    Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic. ... number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions. Key words: Tryptophan ...

  18. Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design

    Directory of Open Access Journals (Sweden)

    Md. Saddam Hossain

    2017-01-01

    Full Text Available Tuberculosis (TB is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using “Allele Frequency Database,” we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

  19. Effects of Surface Epitope Coverage on the Sensitivity of Displacement Assays that Employ Modified Nanoparticles: Using Bisphenol A as a Model Analyte

    Directory of Open Access Journals (Sweden)

    Yang Lu

    2016-08-01

    Full Text Available With the ever-increasing use of nanoparticles in immunosensors, a fundamental study on the effect of epitope density is presented herein, with a small molecule epitope, on the performance of the displacement assay format in an enzyme-linked immunosorbent assay (ELISA. Thiolated bisphenol A (BPA functionalized gold nanoparticles (cysBPAv-AuNPs and specific anti-BPA antibodies are employed for this purpose. It is shown that the displacement of cysBPAv-AuNPs bound to the immobilized antibodies was influenced by both the avidity of bound cysBPAv-AuNPs and the concentration of free BPA to displace it. The importance of surface epitope density was that it changed the number of epitopes in close proximity to the antibody-binding site. This then influenced the avidity of cysBPAv-AuNPs bound to the immobilized antibody. Furthermore, the molar epitope concentration in an assay appears to affect the degree of antibody binding site saturation. Controlling surface epitope density of the functionalized nanoparticles and molar epitope concentration in an assay leads to a decrease of the concentration of free BPA required to displace the bound cysBPAv-AuNP, and hence better assay performance with regards to the D50 value and dynamic range in the displacement assay.

  20. Evaluation and use of in-silico structure-based epitope prediction with foot-and-mouth disease virus.

    Directory of Open Access Journals (Sweden)

    Daryl W Borley

    this epitope prediction method are presented.

  1. The immune epitope database: a historical retrospective of the first decade.

    Science.gov (United States)

    Salimi, Nima; Fleri, Ward; Peters, Bjoern; Sette, Alessandro

    2012-10-01

    As the amount of biomedical information available in the literature continues to increase, databases that aggregate this information continue to grow in importance and scope. The population of databases can occur either through fully automated text mining approaches or through manual curation by human subject experts. We here report our experiences in populating the National Institute of Allergy and Infectious Diseases sponsored Immune Epitope Database and Analysis Resource (IEDB, http://iedb.org), which was created in 2003, and as of 2012 captures the epitope information from approximately 99% of all papers published to date that describe immune epitopes (with the exception of cancer and HIV data). This was achieved using a hybrid model based on automated document categorization and extensive human expert involvement. This task required automated scanning of over 22 million PubMed abstracts followed by classification and curation of over 13 000 references, including over 7000 infectious disease-related manuscripts, over 1000 allergy-related manuscripts, roughly 4000 related to autoimmunity, and 1000 transplant/alloantigen-related manuscripts. The IEDB curation involves an unprecedented level of detail, capturing for each paper the actual experiments performed for each different epitope structure. Key to enabling this process was the extensive use of ontologies to ensure rigorous and consistent data representation as well as interoperability with other bioinformatics resources, including the Protein Data Bank, Chemical Entities of Biological Interest, and the NIAID Bioinformatics Resource Centers. A growing fraction of the IEDB data derives from direct submissions by research groups engaged in epitope discovery, and is being facilitated by the implementation of novel data submission tools. The present explosion of information contained in biological databases demands effective query and display capabilities to optimize the user experience. Accordingly, the

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex t...

  3. Thyroid Autoantibodies Display both “Original Antigenic Sin” and Epitope Spreading

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    Sandra M. McLachlan

    2017-12-01

    Full Text Available Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg, thyroid peroxidase (TPO, and the thyrotropin receptor (TSHR A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of spontaneously arising pathogenic TSHR antibodies in transgenic human thyrotropin receptor/NOD.H2h4 mice by injecting a non-pathogenic form of TSHR A-subunit protein also provides evidence for original antigenic sin. From other studies, antigen presentation by B cells, not dendritic cells, is likely responsible for original antigenic sin. Recognition of restricted epitopes on the large glycosylated thyroid autoantigens (60-kDa A-subunit, 100-kDa TPO, and 600-kDa Tg facilitates exploring the amino acid locations in the immunodominant regions. Epitope spreading has also been revealed by autoantibodies in thyroid autoimmunity. In humans, and in mice that spontaneously develop autoimmunity to all three thyroid autoantigens, autoantibodies develop first to Tg and later to TPO and the TSHR A-subunit. The pattern of intermolecular epitope spreading is related in part to the thyroidal content of Tg, TPO and TSHR A-subunit and to the molecular sizes of these proteins. Importantly, the epitope spreading pattern provides a rationale for future antigen-specific manipulation to block the development of all thyroid autoantibodies by inducing tolerance to Tg, first in the autoantigen cascade. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment, preventing the development of pathogenic TSHR antibodies.

  4. CD4+ T-cell epitope prediction using antigen processing constraints.

    Science.gov (United States)

    Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

    2016-05-01

    T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

    International Nuclear Information System (INIS)

    Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik; Hsu, Catherine; Schneemann, Anette; Manchester, Marianne; Olson, Mark; Reddy, Vijay

    2009-01-01

    Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NΔ52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

  6. Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine

    International Nuclear Information System (INIS)

    Xu Wei; Chu Yiwei; Zhang Ruihua; Xu Huanbin; Wang Ying; Xiong Sidong

    2005-01-01

    CD8 + T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBV-dominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc 18-27 , was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C 18-27 encoding gene. ERTS fusion significantly enhanced specific CD8 + T cell responses in terms of CTL cytolysis as well as IFN-γ secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro

    Directory of Open Access Journals (Sweden)

    Caixia Gao

    2017-12-01

    Full Text Available Swine leukocyte antigen (SLA class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs. Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m using splicing overlap extension polymerase chain reaction (SOE-PCR. The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV and four porcine reproductive and respiratory syndrome virus (PRRSV epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an

  9. Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

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    Anouk C.M. Platteel

    2017-08-01

    Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

  10. Public epitopes and the antigenic structure of the HLA molecules.

    Science.gov (United States)

    Rodey, G E; Fuller, T C

    1987-01-01

    Simplified procedures for determining amino acid sequences in proteins and nucleotide sequences in DNA have rapidly expanded the number of MHC molecules for which primary amino acid structure is known. These molecules will be especially valuable as tools to study the structure-function relationships of globular proteins because of the extensive polymorphism of genes coding the MHC genes products. The general three-dimensional structure of class I MHC molecules was recently deduced, but the more subtle topographical microconformations are still undefined. Definition and topographical mapping of epitopes, defined by serological or cellular immune effector products, will be critical probes for these three-dimensional studies. Comparative studies of amino acid sequences among various MHC and molecules have revealed distinct regions of hypervariability in the alpha-1 and -2 domains of class I heavy chains and the alpha-1 and beta-1 domains of most class II molecules. Mutant MHC molecules that differ from each other by no more than one to three amino acids can have structural changes which may result in a loss of the private epitopes that defined the allelic gene product. On the basis of these studies, the private epitopes are thought to be determined by one or more of the hypervariable regions. Similar studies of the relationships between specific regions of the molecule and public epitopes are not fully explored. Because public epitopes are partially conserved structures, one might expect that their structure is not principally determined by hypervariable region. In fact, however, some public epitopes, such as A2/B17 and BW4/Bw6, do map to diversity regions. Epitope mapping as a means of identifying specific topographic sites and relating these sites to specific functional regions of the molecule will be difficult unless the epitopes themselves are better defined. Thus, the capacity to distinguish spatially distinct public epitopes from cross-reactive homologous

  11. Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity

    DEFF Research Database (Denmark)

    Miller, Yury I; Choi, Soo-Ho; Wiesner, Philipp

    2011-01-01

    are a major target of innate immunity, recognized by a variety of "pattern recognition receptors" (PRRs). By analogy with microbial "pathogen-associated molecular patterns" (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent "danger (or damage......)-associated molecular patterns" (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Furthermore, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation...

  12. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

    Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

    Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families.......Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individual...

  13. An assessment on epitope prediction methods for protozoa genomes

    Directory of Open Access Journals (Sweden)

    Resende Daniela M

    2012-11-01

    Full Text Available Abstract Background Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve performance and a threshold dependent method that employs a confusion matrix; b integrating data from experimentally validated and in silico predicted epitopes; and c integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes. Results A database-driven epitope prediction method was developed with built-in functions that were capable of: a removing experimental data redundancy; b parsing algorithms predictions and storage experimental validated and predict data; and c evaluating algorithm performances. Results show that a better performance is achieved when the combined prediction is considered. This is particularly true for B cell epitope predictors, where the combined prediction of AAP12 and BCPred12 reached an AUC value

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

    Directory of Open Access Journals (Sweden)

    Encheng Sun

    Full Text Available West Nile virus (WNV is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1 of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24 were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV, Newcastle Disease Virus (NDV, Duck Plague Virus (DPV and Goose Parvovirus (GPV antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and

  16. Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

    Science.gov (United States)

    Gardyan, Adriane; Osen, Wolfram; Zörnig, Inka; Podola, Lilli; Agarwal, Maria; Aulmann, Sebastian; Ruggiero, Eliana; Schmidt, Manfred; Halama, Niels; Leuchs, Barbara; von Kalle, Christof; Beckhove, Philipp; Schneeweiss, Andreas; Jäger, Dirk; Eichmüller, Stefan B

    2015-06-01

    Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. © 2014 UICC.

  17. Identification of H-2d Restricted T Cell Epitope of Foot-and-mouth Disease Virus Structural Protein VP1

    Directory of Open Access Journals (Sweden)

    Zhang Zhong-Wang

    2011-09-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious and devastating disease affecting livestock that causes significant financial losses. Therefore, safer and more effective vaccines are required against Foot-and-mouth disease virus(FMDV. The purpose of this study is to screen and identify an H-2d restricted T cell epitope from the virus structural protein VP1, which is present with FMD. We therefore provide a method and basis for studying a specific FMDV T cell epitope. Results A codon-optimized expression method was adopted for effective expression of VP1 protein in colon bacillus. We used foot-and-mouth disease standard positive serum was used for Western blot detection of its immunogenicity. The VP1 protein was used for immunizing BALB/c mice, and spleen lymphocytes were isolated. Then, a common in vitro training stimulus was conducted for potential H-2Dd, H-2Kd and H-2Ld restricted T cell epitope on VP1 proteins that were predicted and synthesized by using a bioinformatics method. The H-2Kd restricted T cell epitope pK1 (AYHKGPFTRL and the H-2Dd restricted T cell epitope pD7 (GFIMDRFVKI were identified using lymphocyte proliferation assays and IFN-γ ELISPOT experiments. Conclusions The results of this study lay foundation for studying the FMDV immune process, vaccine development, among other things. These results also showed that, to identify viral T cell epitopes, the combined application of bioinformatics and molecular biology methods is effective.

  18. Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Leticia Barboza Rocha

    2017-10-01

    Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

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

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    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.

  20. An Approach for a Synthetic CTL Vaccine Design against Zika Flavivirus Using Class I and Class II Epitopes Identified by Computer Modeling

    Directory of Open Access Journals (Sweden)

    Edecio Cunha-Neto

    2017-06-01

    Full Text Available The threat posed by severe congenital abnormalities related to Zika virus (ZKV infection during pregnancy has turned development of a ZKV vaccine into an emergency. Recent work suggests that the cytotoxic T lymphocyte (CTL response to infection is an important defense mechanism in response to ZKV. Here, we develop the rationale and strategy for a new approach to developing cytotoxic T lymphocyte (CTL vaccines for ZKV flavivirus infection. The proposed approach is based on recent studies using a protein structure computer model for HIV epitope selection designed to select epitopes for CTL attack optimized for viruses that exhibit antigenic drift. Because naturally processed and presented human ZKV T cell epitopes have not yet been described, we identified predicted class I peptide sequences on ZKV matching previously identified DNV (Dengue class I epitopes and by using a Major Histocompatibility Complex (MHC binding prediction tool. A subset of those met the criteria for optimal CD8+ attack based on physical chemistry parameters determined by analysis of the ZKV protein structure encoded in open source Protein Data File (PDB format files. We also identified candidate ZKV epitopes predicted to bind promiscuously to multiple HLA class II molecules that could provide help to the CTL responses. This work suggests that a CTL vaccine for ZKV may be possible even if ZKV exhibits significant antigenic drift. We have previously described a microsphere-based CTL vaccine platform capable of eliciting an immune response for class I epitopes in mice and are currently working toward in vivo testing of class I and class II epitope delivery directed against ZKV epitopes using the same microsphere-based vaccine.

  1. Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2013-08-01

    Full Text Available J F Alderete, Calvin J NeaceSchool of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USAAbstract: There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI. Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD, α-enolase (ENO, and glyceraldehyde-3-phosphate dehydrogenase (GAP. We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera. We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA, dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis.Keywords: diagnostics, point-of-care, targets, trichomonosis

  2. Antibody response to a synthetic peptide covering a LAV-1/HTLV-IIIB neutralization epitope and disease progression

    NARCIS (Netherlands)

    Boucher, C. A.; de Wolf, F.; Houweling, J. T.; Bakker, M.; Dekker, J.; Roos, M. T.; Coutinho, R. A.; van der Noordaa, J.; Goudsmit, J.

    1989-01-01

    Sequential sera of homosexual men participating in a prospective study on the incidence of HIV-1 infection and risk factors for AIDS were tested for the presence of antibodies to a synthetic 17-mer (Neu21; KSIRIQRGPGRAFVTIG) representing a neutralization epitope as present on the LAV-1/HTLV-IIIB

  3. The Occurrence of Sequences Identical with Epitopes from the Allergen Pen a 1.0102 Among Food and Non-Food Proteins

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    Minkiewicz Piotr

    2015-03-01

    Full Text Available The presence of common epitopes among tropomyosins of invertebrates, including arthropods, e.g. edible ones, may help to explain the molecular basis of cross-reactivity between allergens. The work presented is the first survey concerning global distribution of epitopes from Pen a 1.0102 in universal proteome. In the group of known tropomyosin epitopes, the fragment with the sequence ESKIVELEEEL was found in the sequence of channel catfish (Ictalurus punctatus tropomyosin. To date, this is the first result suggesting the presence of a complete sequential epitope interacting with gE in vertebrate tropomyosin. Another fragment with the sequence VAALNRRIQL, a major part of the epitope, was found in 11 fish, 8 amphibians, 3 birds, 19 mammalians and 4 human tropomyosin sequences. Identical epitopes are common in sequences of invertebrate tropomyosins, including food and non-food allergens annotated in the Allergome database. The rare pentapeptide with the DEERM sequence occurs in proteins not sharing homology with tropomyosins. Pathogenic microorganisms are the most abundant category of organisms synthesizing such proteins.

  4. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

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    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  5. Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

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    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

    Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

  6. Epitope analysis of anti-myeloperoxidase antibodies in patients with ANCA-associated vasculitis.

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    Shen-Ju Gou

    Full Text Available OBJECTIVE: Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA directing myeloperoxidase (MPO in ANCA-associated vasculitis (AAV. The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV. METHODS: Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands. RESULTS: Sera from 45 of the 77 (58.4% patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset. CONCLUSION: The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis.

  7. The immune epitope database (IEDB) 3.0

    Science.gov (United States)

    Vita, Randi; Overton, James A.; Greenbaum, Jason A.; Ponomarenko, Julia; Clark, Jason D.; Cantrell, Jason R.; Wheeler, Daniel K.; Gabbard, Joseph L.; Hix, Deborah; Sette, Alessandro; Peters, Bjoern

    2015-01-01

    The IEDB, www.iedb.org, contains information on immune epitopes—the molecular targets of adaptive immune responses—curated from the published literature and submitted by National Institutes of Health funded epitope discovery efforts. From 2004 to 2012 the IEDB curation of journal articles published since 1960 has caught up to the present day, with >95% of relevant published literature manually curated amounting to more than 15 000 journal articles and more than 704 000 experiments to date. The revised curation target since 2012 has been to make recent research findings quickly available in the IEDB and thereby ensure that it continues to be an up-to-date resource. Having gathered a comprehensive dataset in the IEDB, a complete redesign of the query and reporting interface has been performed in the IEDB 3.0 release to improve how end users can access this information in an intuitive and biologically accurate manner. We here present this most recent release of the IEDB and describe the user testing procedures as well as the use of external ontologies that have enabled it. PMID:25300482

  8. Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies.

    Science.gov (United States)

    Mourad, W; Bernier, D; Jobin, M; Hébert, J

    1989-11-01

    Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

  9. HIV-1 subtype A gag variability and epitope evolution.

    Science.gov (United States)

    Abidi, Syed Hani; Kalish, Marcia L; Abbas, Farhat; Rowland-Jones, Sarah; Ali, Syed

    2014-01-01

    The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

  10. HIV-1 subtype A gag variability and epitope evolution.

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    Syed Hani Abidi

    Full Text Available OBJECTIVE: The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. METHODS: We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. RESULTS: The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. CONCLUSION: It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

  11. Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

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    Gao Fan

    2012-01-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Science.gov (United States)

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

    2018-01-01

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

  14. Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

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    Annette Fink

    2014-02-01

    Full Text Available Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I glycoproteins, are often identified by “reverse immunology”, a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E phase protein, the m164 epitope is presented already during the Immediate Early (IE phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

  15. Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein.

    Science.gov (United States)

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

    2012-05-01

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

  16. High Throughput T Epitope Mapping and Vaccine Development

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    Giuseppina Li Pira

    2010-01-01

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

  17. Expression, purification and epitope analysis of Pla a 2 allergen from Platanus acerifolia pollen.

    Science.gov (United States)

    Wang, De-Wang; Ni, Wei-Wei; Zhou, Yan-Jun; Huang, Wen; Cao, Meng-Da; Meng, Ling; Wei, Ji-Fu

    2018-01-01

    Platanus acerifolia is one of the major sources of outdoor allergens to humans, and can induce allergic asthma, rhinitis, dermatitis and other allergic diseases. Pla a 2 is a polygalacturonase and represents the major allergen identified in P. acerifolia pollen. The aim of the present study was to express and purify Pla a 2, and to predict B and T cell epitopes of Pla a 2. The gene encoding Pla a 2 was cloned into the pET28a vector and subsequently transfected into ArcticExpress™ (DE3) Escherichia coli cells; purified Pla a 2 was analyzed by western blot analysis. The results of the present study revealed that the Pla a 2 allergen has the ability to bind immunoglobulin E within the sera of patients allergic to P. acerifolia pollen. In addition, the B cell epitopes of Pla a 2 were predicted using the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides and BepiPred 1.0 software; T cell epitopes were predicted using NetMHCIIpan ‑3.0 and ‑2.2. In total, eight B cell epitopes (15‑24, 60‑66, 78‑86, 109‑124, 232‑240, 260‑269, 298‑306 and 315‑322) and five T cell epitopes (62‑67, 86‑91, 125‑132, 217‑222 and 343‑350) were predicted in the present study. These findings may be used to improve allergen immunotherapies and reduce the frequency of pollen‑associated allergic reactions.

  18. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

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    Werner Smidt

    Full Text Available The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1 infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS. Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  19. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Science.gov (United States)

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  20. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

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    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  1. Mapping the epitopes of a neutralizing antibody fragment directed against the lethal factor of Bacillus anthracis and cross-reacting with the homologous edema factor.

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    Philippe Thullier

    Full Text Available The lethal toxin (LT of Bacillus anthracis, composed of the protective antigen (PA and the lethal factor (LF, plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF to form the edema toxin (ET, which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236, of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260 was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

  2. Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

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    Yaakov Maman

    2011-10-01

    Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

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

    Science.gov (United States)

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

    2018-04-02

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

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein

    Directory of Open Access Journals (Sweden)

    Kiener Tanja K

    2012-02-01

    Full Text Available Abstract Background Enterovirus 71 (EV71 has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. Results We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. Conclusions Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non

  6. Neutralization epitopes on HIV pseudotyped with HTLV-I: Conservation of carbohydrate Epitopes

    DEFF Research Database (Denmark)

    Sørensen, A M; Nielsen, C; Arendrup, M

    1994-01-01

    One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4......-negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility...... by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes....

  7. Neutralization epitopes on HIV pseudotyped with HTLV-I: conservation of carbohydrate epitopes

    DEFF Research Database (Denmark)

    Sørensen, A M; Nielsen, C; Arendrup, M

    1994-01-01

    One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4......-negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility...... by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes....

  8. Prediction of residues in discontinuous B-cell epitopes using protein 3D structures

    DEFF Research Database (Denmark)

    Andersen, P.H.; Nielsen, Morten; Lund, Ole

    2006-01-01

    . We show that the new structure-based method has a better performance for predicting residues of discontinuous epitopes than methods based solely on sequence information, and that it can successfully predict epitope residues that have been identified by different techniques. DiscoTope detects 15...... experimental epitope mapping in both rational vaccine design and development of diagnostic tools, and may lead to more efficient epitope identification....

  9. File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Oth.Liv.10.Epitope_tags.AllCell [Chip-atlas[Archive

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  18. File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

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  6. File list: Oth.YSt.50.Epitope_tags.AllCell [Chip-atlas[Archive

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  7. File list: Oth.PSC.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: Oth.Oth.10.Epitope_tags.AllCell [Chip-atlas[Archive

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  9. File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

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  12. File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

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  13. File list: Oth.Epd.20.Epitope_tags.AllCell [Chip-atlas[Archive

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  1. In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

    DEFF Research Database (Denmark)

    Moise, Leonard; McMurry, Julie A; Buus, Søren

    2009-01-01

    Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...

  2. Identification and characterization of survivin-derived H-2Kb-restricted CTL epitopes

    DEFF Research Database (Denmark)

    Hofmann, Uta B; Voigt, Heike; Andersen, Mads H

    2009-01-01

    for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes...

  3. Use of two predictive algorithms of the world wide web for the identification of tumor-reactive T-cell epitopes.

    Science.gov (United States)

    Lu, J; Celis, E

    2000-09-15

    Tumor cells can be effectively recognized and eliminated by CTLs. One approach for the development of CTL-based cancer immunotherapy for solid tumors requires the use of the appropriate immunogenic peptide epitopes that are derived from defined tumor-associated antigens. Because CTL peptide epitopes are restricted to specific MHC alleles, to design immune therapies for the general population it is necessary to identify epitopes for the most commonly found human MHC alleles. The identification of such epitopes has been based on MHC-peptide-binding assays that are costly and labor-intensive. We report here the use of two computer-based prediction algorithms, which are readily available in the public domain (Internet), to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). These algorithms identified three candidate peptides that we studied for their capacity to induce CTL responses in vitro using lymphocytes from HLA-B7+ normal blood donors. The results show that one of these peptides, CEA9(632) (IPQQHTQVL) was efficient in the induction of primary CTL responses when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that express HLA-B7 and produce CEA. The identification of this HLA-B7-restricted CTL epitope will be useful for the design of ethnically unbiased, widely applicable immunotherapies for common solid epithelial tumors expressing CEA. Moreover, our strategy of identifying MHC class I-restricted CTL epitopes without the need of peptide/HLA-binding assays provides a convenient and cost-saving alternative approach to previous methods.

  4. Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague.

    Science.gov (United States)

    Zvi, Anat; Rotem, Shahar; Zauberman, Ayelet; Elia, Uri; Aftalion, Moshe; Bar-Haim, Erez; Mamroud, Emanuelle; Cohen, Ofer

    2017-10-20

    The causative agent of Plague, Yersinia pestis, is a highly virulent pathogen and a potential bioweapon. Depending on the route of infection, two prevalent occurrences of the disease are known, bubonic and pneumonic. The latter has a high fatality rate. In the absence of a licensed vaccine, intense efforts to develop a safe and efficacious vaccine have been conducted, and humoral-driven subunit vaccines containing the F1 and LcrV antigens are currently under clinical trials. It is well known that a cellular immune response might have an essential additive value to immunity and protection against Y. pestis infection. Nevertheless, very few documented epitopes eliciting a protective T-cell response have been reported. Here, we present a combined high throughput computational and experimental effort towards identification of CD8 T-cell epitopes. All 4067 proteins of Y. pestis were analyzed with state-of-the-art recently developed prediction algorithms aimed at mapping potential MHC class I binders. A compilation of the results obtained from several prediction methods revealed a total of 238,000 peptide candidates, which necessitated downstream filtering criteria. Our previously established and proven approach for enrichment of true positive CTL epitopes, which relies on mapping clusters rich in tandem or overlapping predicted MHC binders ("hotspots"), was applied, as well as considerations of predicted binding affinity. A total of 1532 peptides were tested for their ability to elicit a specific T-cell response by following the production of IFNγ from splenocytes isolated from vaccinated mice. Altogether, the screen resulted in 178 positive responders (11.8%), all novel Y. pestis CTL epitopes. These epitopes span 113 Y. pestis proteins. Substantial enrichment of membrane-associated proteins was detected for epitopes selected from hotspots of predicted MHC binders. These results considerably expand the repertoire of known CTL epitopes in Y. pestis and pave the way to

  5. Comparative characteristic of the methods of protein antigens epitope mapping

    Directory of Open Access Journals (Sweden)

    O. Yu. Galkin

    2014-08-01

    Full Text Available Comparative analysis of experimental methods of epitope mapping of protein antigens has been carried out. The vast majority of known techniques are involved in immunochemical study of the interaction of protein molecules or peptides with antibodies of corresponding specifici­ty. The most effective and widely applicable metho­dological techniques are those that use synthetic and genetically engineered peptides. Over the past 30 years, these groups of methods have travelled a notable evolutionary path up to the maximum automation and the detection of antigenic determinants of various types (linear and conformational epitopes, and mimotopes. Most of epitope searching algorithms were integrated into a computer program, which greatly facilitates the analysis of experimental data and makes it possible to create spatial models. It is possible to use comparative epitope mapping for solving the applied problems; this less time-consuming method is based on the analysis of competition between different antibodies interactions with the same antigen. The physical method of antigenic structure study is X-ray analysis of antigen-antibody complexes, which may be applied only to crystallizing­ proteins, and nuclear magnetic resonance.

  6. High-throughput epitope profiling of snake venom toxins

    DEFF Research Database (Denmark)

    Engmark, Mikael; Andersen, Mikael Rørdam; Laustsen, Andreas Hougaard

    Insight into the molecular details of polyclonal antivenom antibody specificity is a prerequisite for accurate prediction of cross-reactivity and can provide a basis for design of novel antivenoms. In this work, a highthroughput approach was applied to characterize linear elements in epitopes in ...... toxins from four African mamba and three neurotoxic cobra snakes obtained from public databases....

  7. Bioinformatics Analysis of Envelope Glycoprotein E epitopes of ...

    African Journals Online (AJOL)

    The E glycoprotein of dengue virus is responsible for the viral binding to the receptor. The crystal structure of envelope glycoprotein has already been determined. However, where the well-defined Bcell and T-cell epitopes are located is still a question. Because of the large variations among the four dengue genotypes, it is ...

  8. Indirect recognition of HLA epitopes in solid organ transplantation

    NARCIS (Netherlands)

    Geneugelijk, C.C.A.

    2017-01-01

    Alloreactivity due to HLA mismatches between donor and recipient remains the major limiting factor in successful graft outcome after solid organ transplantation. However, the immunogenicity of individual HLA mismatches is highly variable. Therefore, epitope-based HLA matching may be a sophisticated

  9. Catalase epitopes vaccine design for Helicobacter pylori : A ...

    African Journals Online (AJOL)

    Catalase, an important enzyme in the virulence of H. pylori, could be a suitable candidate for vaccine design because it is highly conserved, which is important for the survival of H. pylori; it is expressed in high level and it is exposed on the surface of the bacteria. In this study, we designed epitope-based vaccine for catalase ...

  10. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    Science.gov (United States)

    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. © 2016 The Histochemical Society.

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

    Science.gov (United States)

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

    2017-05-01

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

  12. Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

    Science.gov (United States)

    Powell, Rebecca L R; Totrov, Maxim; Itri, Vincenza; Liu, Xiaomei; Fox, Alisa; Zolla-Pazner, Susan

    2017-09-01

    We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4β7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens. IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine

  13. Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

    Science.gov (United States)

    Panayotatos, N; Radziejewska, E; Acheson, A; Somogyi, R; Thadani, A; Hendrickson, W A; McDonald, N Q

    1995-06-09

    By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

  14. Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Edgar E. Lara-Ramírez

    2013-11-01

    Full Text Available The past pandemic strain H1N1 (A (H1N1pdm09 has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI database were used for the analysis. The epitope compositions in A (H1N1pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB, were compared at genomic level. The result showed that A (H1N1 pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA, which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1pdm09 virus. This study may be helpful for better understanding of A (H1N1pdm09 and the production of new influenza vaccines.

  15. Improving Multi-Epitope Long Peptide Vaccine Potency by Using a Strategy that Enhances CD4+ T Help in BALB/c Mice.

    Directory of Open Access Journals (Sweden)

    Haniyeh Ghaffari-Nazari

    Full Text Available Peptide-based vaccines are attractive approaches for cancer immunotherapy; but the success of these vaccines in clinical trials have been limited. Our goal is to improve immune responses and anti-tumor effects against a synthetic, multi-epitope, long peptide from rat Her2/neu (rHer2/neu using the help of CD4+ T cells and appropriate adjuvant in a mouse tumor model. Female BALB/c mice were vaccinated with P5+435 multi-epitope long peptide that presents epitopes for cytotoxic T lymphocytes (CTL in combination with a universal Pan DR epitope (PADRE or CpG-oligodeoxynucleotides (CpG-ODNs as a Toll-like receptor agonist adjuvant. The results show that vaccination with the multi-epitope long peptide in combination with the PADRE peptide and CpG-ODN induced expansion of subpopulations of CD4+ and CD8+ cells producing IFN-γ, the average tumor size in the vaccinated mice was less than that of the other groups, and tumor growth was inhibited in 40% of the mice in the vaccinated group. The mean survival time was 82.6 ± 1.25 days in mice vaccinated with P5+435 + CpG+ PADRE. Our results demonstrate that inclusion of PADRE and CpG with the peptide vaccine enhanced significant tumor specific-immune responses in vaccinated mice.

  16. Polymorphism of 41 kD Flagellin Gene and Its Human B-Cell Epitope in Borrelia burgdorferi Strains of China

    Directory of Open Access Journals (Sweden)

    Huixin Liu

    2016-01-01

    Full Text Available The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease.

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

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

    Full Text Available 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.

  18. Levels of HIV1 gp120 3D B-cell epitopes mutability and variability: searching for possible vaccine epitopes.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich

    2010-01-01

    We used a DiscoTope 1.2 (http://www.cbs.dtu.dk/services/DiscoTope/), Epitopia (http://epitopia.tau.ac.il/) and EPCES (http://www.t38.physik.tu-muenchen.de/programs.htm) algorithms to map discontinuous B-cell epitopes in HIV1 gp120. The most mutable nucleotides in HIV genes are guanine (because of G to A hypermutagenesis) and cytosine (because of C to U and C to A mutations). The higher is the level of guanine and cytosine usage in third (neutral) codon positions and the lower is their level in first and second codon positions of the coding region, the more stable should be an epitope encoded by this region. We compared guanine and cytosine usage in regions coding for five predicted 3D B-cell epitopes of gp120. To make this comparison we used GenBank resource: 385 sequences of env gene obtained from ten HIV1-infected individuals were studied (http://www.barkovsky.hotmail.ru/Data/Seqgp120.htm). The most protected from nonsynonymous nucleotide mutations of guanine and cytosine 3D B-cell epitope is situated in the first conserved region of gp120 (it is mapped from 66th to 86th amino acid residue). We applied a test of variability to confirm this finding. Indeed, the less mutable predicted B-cell epitope is the less variable one. MEGA4 (standard PAM matrix) was used for the alignments and "VVK Consensus" algorithm (http://www.barkovsky.hotmail.ru) was used for the calculations.

  19. The neo-epitope specific PRO-C3 ELISA measures true formation of type III collagen associated with liver and muscle parameters

    DEFF Research Database (Denmark)

    Nielsen, Mette J; Nedergaard, Anders F; Sun, Shu

    2013-01-01

    AIM: The present study describes the assessment of true formation of type III collagen in different pathologies using a neo-epitope specific competitive Enzyme-linked immunosorbent assay (ELISA) towards the N-terminal propeptide of type III collagen (PRO-C3). METHODS: The monoclonal antibody...... was raised against the N-protease mediated cleavage site of the N-terminal propeptide of type III collagen and a competitive ELISA was developed using the selected antibody. The assay was evaluated in relation to neo-epitope specificity, technical performance, and as a marker for liver fibrosis and muscle...... mass using the rat carbon tetrachloride (CCl4) model and a study of immobilization induced muscle loss in humans, respectively. RESULTS: The ELISA was neo-epitope specific, technically stable and can be assessed in serum and plasma samples. In the CCl4 liver fibrosis model it was observed that serum...

  20. Loss of Anti-Viral Immunity by Infection with a Virus Encoding a Cross-Reactive Pathogenic Epitope

    OpenAIRE

    Chen, Alex T.; Cornberg, Markus; Gras, Stephanie; Guillonneau, Carole; Rossjohn, Jamie; Trees, Andrew; Emonet, Sebastien; de la Torre, Juan C.; Welsh, Raymond M.; Selin, Liisa K.

    2012-01-01

    Author Summary The purpose of vaccination against viruses is to induce strong neutralizing antibody responses that inactivate viruses on contact and strong T cell responses that attack and kill virus-infected cells. Some viruses, however, like HIV and hepatitis C virus, are only weakly controlled by neutralizing antibody, so T cell immunity is very important for control of these infections. T cells recognize small virus-encoded peptides, called epitopes, presented on the surface of infected c...

  1. The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.

    Directory of Open Access Journals (Sweden)

    Siri Dørum

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

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

    Science.gov (United States)

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

    2010-01-01

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

  3. Therapeutic Vaccine Against HIV, Viral Variability, Cytotoxic T Lymphocyte Epitopes, and Genetics of Patients.

    Science.gov (United States)

    Fleury, Herve; Tumiotto, Camille; Bellecave, Pantxika; Recordon-Pinson, Patricia

    2018-01-01

    The scientific and medical community is seeking to cure HIV. Several pathways have been or are being explored including therapeutic vaccination. Viroimmunological studies on primary infection as well as on elite controllers have demonstrated the importance of the cytotoxic CD8 response and have mainly oriented research on vaccine constructs toward this type of response. The results of these trials are clearly not commensurate with the hope placed in them. Might there be one or more uncontrolled variables? The genetics of patients need to be taken into consideration, especially their human lymphocyte antigen (HLA) alleles. There is a need to find a balance between the conservation of cytotoxic T lymphocyte (CTL) epitopes and presentation by HLA alleles. The pathway is a narrow one between adaptation of the virus to HLA I restriction and the definition of conserved proviral CTL epitopes presentable by HLA I alleles. It is likely that the genetics of patients will need to be considered for HIV-1 vaccine studies and that multidisciplinary collaboration will be essential in this field of infectious diseases.

  4. On the synthesis of peptide imprinted polymers by a combined suspension-Epitope polymerization method

    International Nuclear Information System (INIS)

    Kotrotsiou, O.; Chaitidou, S.; Kiparissides, C.

    2009-01-01

    In the past, molecularly imprinted polymers (MIPs), prepared by free-radical bulk polymerization, have been used for the selective recognition of small biomolecules (i.e., amino acids and amino acid derivatives). Presently, there is a need for the synthesis of MIPs capable of recognizing larger biomolecules (i.e., peptides and proteins). Moreover, it is highly desirable the production of MIP microparticles with well-defined morphological characteristics (e.g., particle size distribution, porosity, etc.) via particulate polymerization techniques. In the present study, the synthesis of molecularly imprinted microparticles, produced via the suspension and inverse suspension polymerization methods, using the 'epitope approach', is reported. The hydrophobic (i.e., Boc-Trp-Trp-Trp) or hydrophilic (i.e., His-Phe) oligo-peptides were employed as template molecules. The potential of the combined suspension polymerization method with the 'epitope approach' for the production of MIP microparticles is demonstrated, as well as the specificity and selectivity characteristics of the MIP microparticles towards hydrophobic and hydrophilic oligo-peptides. The proposed method appears to be a very promising and efficient technique for separation of proteins.

  5. 'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

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    Nathali Kaushansky

    Full Text Available Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE associated with a single ("classical" or multiple ("complex" anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells. Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for

  6. The Cancer Exome Generated by Alternative mRNA Splicing Dilutes Predicted HLA Class I Epitope Density

    DEFF Research Database (Denmark)

    Stranzl, Thomas; Larsen, Mette Voldby; Lund, Ole

    2012-01-01

    Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes...... is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based...... on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I...

  7. Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape.

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    Jens Dinter

    2015-03-01

    Full Text Available Dendritic cells (DCs and macrophages (Møs internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL. However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

  8. Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies.

    Science.gov (United States)

    Mourad, W; Mécheri, S; Peltre, G; David, B; Hébert, J

    1988-11-15

    The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.

  9. Programmed Death-1 expression on Epstein Barr virus specific CD8+ T cells varies by stage of infection, epitope specificity, and T-cell receptor usage.

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    Thomas C Greenough

    Full Text Available BACKGROUND: Programmed Death-1 (PD-1 is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM and convalescence. METHODOLOGY/PRINCIPAL FINDINGS: Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage. CONCLUSIONS/SIGNIFICANCE: These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage.

  10. Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in spike 1 domain and membrane protein of feline infectious peritonitis virus.

    Science.gov (United States)

    Takano, Tomomi; Morioka, Hiroyuki; Gomi, Kohji; Tomizawa, Keisuke; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2014-04-01

    Feline infectious peritonitis virus (FIP virus: FIPV) causes a fatal disease in wild and domestic cats. The development of an FIP-preventive vaccine requires an antigen that does not induce antibody-dependent enhancement, and T helper (Th)1 activity plays an important role in protect against FIPV infection. In the present study, we identified synthetic peptides including Th1 and a linear immunodominant antibody-binding epitope in the S1 domain and M protein of FIPV. We also identified peptides that strongly induce Th1 activity from those derived from the structural proteins (S, M, and N proteins) of FIPV based on this and previous studies (Satoh et al. [19]). No Th1 epitope-containing peptide was identified in the peptides derived from the S1 domain of type I FIPV. In contrast, 7 Th1 epitope-containing peptides were identified in the S1 domain of type II FIPV, and no linear immunodominant antibody-binding epitope was contained in any of these peptides. Eleven Th1 epitope-containing peptides common to each serotype were identified in the M protein-derived peptides, and 2 peptides (M-11 and M-12) contained the linear immunodominant antibody-binding epitope. Of the peptides derived from the S, M, and N proteins of FIPV, those that induced significantly stronger Th1 activity than that of the FIPV antigen were rescreened, and 4 peptides were identified. When 3 of these peptides (M-9, I-S2-15, and II-S1-24) were selected and administered with CpG-ODNs to SPF cats, M-9 and II-S1-24 induced Th1 activity. Our results may provide important information for the development of a peptide-based vaccine against FIPV infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection.

    Science.gov (United States)

    Richards, Katherine A; DiPiazza, Anthony T; Rattan, Ajitanuj; Knowlden, Zackery A G; Yang, Hongmei; Sant, Andrea J

    2018-01-01

    One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.

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

    Science.gov (United States)

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

    2017-02-28

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

  13. Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

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    Anton M Sholukh

    Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

  14. Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

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    Joos Thomas

    2010-06-01

    Full Text Available Abstract Background Mass spectrometry (MS based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. Results We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. Conclusions For small datasets (a few hundred proteins it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

  15. Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.

    Science.gov (United States)

    Planatscher, Hannes; Supper, Jochen; Poetz, Oliver; Stoll, Dieter; Joos, Thomas; Templin, Markus F; Zell, Andreas

    2010-06-25

    Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

  16. Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin.

    Science.gov (United States)

    Chang, Yao-Wen; Kaneko, Mika K; Yamada, Shinji; Kato, Yukinari

    2018-02-02

    The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope of cPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4) of cPDPN are responsible for the binding of PMab-52 to cPDPN.

  17. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. B-CELL EPITOPE ON THE U1 SNRNP-C AUTOANTIGEN CONTAINS A SEQUENCE SIMILAR TO THAT OF THE HERPES-SIMPLEX VIRUS PROTEIN

    NARCIS (Netherlands)

    MISAKI, Y; YAMAMOTO, K; YANAGI, K; MIURA, H; ICHIJO, H; KATO, T; MATO, T; WELLINGWESTER, S; NISHIOKA, K; ITO, K

    The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the

  19. Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1

    DEFF Research Database (Denmark)

    Barfod, Lea; Dalgaard, Michael B; Pleman, Suzan T

    2011-01-01

    1 (PfEMP1) family is central to both. Here, we present evidence of a P. falciparum evasion mechanism not previously documented: the masking of PfEMP1-specific IgG epitopes by nonspecific IgM. Nonspecific IgM binding to erythrocytes infected by parasites expressing the PfEMP1 protein VAR2CSA...

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

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    José Esteban Muñoz-Medina

    2015-01-01

    Full Text Available 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.

  1. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

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    Daniel J Hui

    Full Text Available Adeno-associated virus (AAV has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC class I epitopes for common human leukocyte antigen (HLA types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  2. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

    Science.gov (United States)

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  3. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay.

    Science.gov (United States)

    Bottino, Carolina G; Gomes, Luciano P; Pereira, José B; Coura, José R; Provance, David William; De-Simone, Salvatore G

    2013-12-03

    The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could

  4. Quantitative Analysis of the Association Angle between T-cell Receptor Vα/Vβ Domains Reveals Important Features for Epitope Recognition.

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    Thomas Hoffmann

    2015-07-01

    Full Text Available T-cell receptors (TCR play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR, which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation.

  5. Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine

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    Widodo

    2018-03-01

    Full Text Available Background: Epstein-Barr virus (EBV can cause cancer in people from around the world. There is no EBV vaccine available for use on a global scale. However, emerging evidence suggests that the epitope on the gp350/220 capsid protein may be developed into an EBV vaccine. Nevertheless, the production of small, single epitope is challenging of stability issues and possible alteration of peptide structure. In this study, a tandem epitope was developed consisting of three single epitopes, aimed to improve stability, antigenicity and preserve epitope structure. Materials and methods: A tandem epitope was designed using bioinformatics based on the epitope structure of the gp350/220 protein. The tandem epitope structure was analyzed using a protein folding method with Abalone software, which was further refined via YASARA force field and molecular repairing using a FoldX method. Immunogenicity was examined with Epitopia software, whereas allergen properties were tested using AlgPred. The pattern of the tandem epitope binding with anti-gp350/220 antibodies was performed using Z-dock and snugDock. The tandem epitope was then overproduced in E. coli strain BL21 as a host cell. Result: Our model demonstrated a successfully designed and overproduced tandem epitope. The tandem epitope demonstrated a similar structure compared with the epitope of whole protein gp350/220. Our epitope also demonstrated non-allergen and antigenicity properties, and possessed antibody binding patterns consistent with whole protein gp350/220. Conclusion and recommendation: These data suggest a novel tandem epitope composed of three similar epitopes demonstrates antigenicity, structure, and binding properties consistent with whole protein gp350/220. We also demonstrate successful production of the tandem epitope using E. coli strain BL21 as a host. Future in vivo experimental animal research is necessary to test the ability of this tandem epitope to stimulate antibody production

  6. HIV p24 as scaffold for presenting conformational HIV Env antigens.

    Directory of Open Access Journals (Sweden)

    Maria Tagliamonte

    Full Text Available Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4(+ and CD8(+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.

  7. In silico epitope prediction, expression and functional analysis of Per a 10 allergen from the American cockroach

    Science.gov (United States)

    Tong, Xunliang; Guo, Miao; Jin, Min; Chen, Hao; Li, Yanming; Wei, Ji-Fu

    2016-01-01

    Cockroach (CR) allergies caused by the American cockroach hyave been recognized to be repsonsible for IgE-mediated type I hypersensitivity worldwide. Per a 10 is one of the recognized main allergens of the American CR. In a previous study, we examined another American CR allergen, Per a 9 in patients with CR allergies and examined epitope sequences in this allergen. In the present study, we aimed to examine epitope sequences in the Per a 10 allergen. for this purpose, the Per a 10 gene was cloned and expressed in Escherichia coli (E. coli) systems. Our results revealed that 9 out of 16 (56.3%) sera from patients with American CR allergies reacted to Per a10, as assessed by ELISA, confirming that Per a 10 is a major allergen of the American CR. Our results also revealed that the expression of CD63 and CCR3 on passively sensitized basophils (obtained sera of patients with American CR allergies) was increased by approximately 2.3-fold, indicating that recombinant Per a 10 is functionally active. In addition, 3 immunoinformatics tools, namely the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the peptides and the results revealed 8 peptides (2–12, 55–67, 98–120, 125–133, 149–160, 170–182, 201–208 and 223–227) as potential B cell epitopes of the Per a 10 allergen. Moreover, Per a 10 was predicted to have 3 T cell epitope sequences, namely 83–92, 139–147 and 162–170. The findings of our study on the CR allergen may prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of CR allergies. PMID:27840898

  8. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  9. Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach

    International Nuclear Information System (INIS)

    Amniai, Laziza; Lippens, Guy; Landrieu, Isabelle

    2011-01-01

    Highlights: → pThr231 of the Tau protein is necessary for the binding of the AT180 antibody. → pSer235 of the Tau protein does not interfere with the AT180 recognition of pThr231. → Epitope mapping is efficiently achieved by combining NMR and FRET spectroscopy. -- Abstract: We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer's disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Foerster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30 nM. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding.

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

    Science.gov (United States)

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

    1996-07-01

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

  11. Emulsified phosphatidylserine, simple and effective peptide carrier for induction of potent epitope-specific T cell responses.

    Directory of Open Access Journals (Sweden)

    Toru Ichihashi

    Full Text Available BACKGROUND: To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL induction capability. METHODOLOGY/PRINCIPAL FINDINGS: Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c(+CD11b(+MHCII(+ conventional dendritic cells (cDCs compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo. CONCLUSIONS/SIGNIFICANCE: This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.

  12. Emulsified phosphatidylserine, simple and effective peptide carrier for induction of potent epitope-specific T cell responses.

    Science.gov (United States)

    Ichihashi, Toru; Satoh, Toshifumi; Sugimoto, Chihiro; Kajino, Kiichi

    2013-01-01

    To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability. Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c(+)CD11b(+)MHCII(+) conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo. This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.

  13. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    International Nuclear Information System (INIS)

    Oh, Sang Kon; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A.

    2006-01-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8 + T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8 + T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K b and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection

  14. Epitopes recognized by CBV4 responding T cells: effect of type 1 diabetes and associated HLA-DR-DQ haplotypes

    International Nuclear Information System (INIS)

    Marttila, Jane; Hyoety, Heikki; Naentoe-Salonen, Kirsti; Simell, Olli; Ilonen, Jorma

    2004-01-01

    The present study aimed at characterizing the epitopes recognized by coxsackievirus B4 (CBV4)-specific T-cell lines established from 23 children with type 1 diabetes (T1D) and 29 healthy children with T1D risk-associated HLA genotypes. Responsiveness to VP1 region was dependent on the specific infection history as 55% of the T-cell lines from donors with neutralizing antibodies to CBV serotypes responded to VP1 peptides compared to none of the T-cell lines from other donors (P = 0.01). The pattern of recognized peptides was dependent of the HLA genotype. Forty-two percent of the T-cell lines from donors carrying the HLA-(DR4)-DQB1*0302 haplotype responded to VP1 peptides 71-80 compared to none of the T-cell lines from donors without this haplotype (P = 0.02). No evidence for the existence of diabetes-specific epitopes was found. Only few epitopes were exclusive recognized by T cells from diabetic children, and in each case only one or two T-cell lines were responding

  15. Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein

    DEFF Research Database (Denmark)

    Ozkokmen, D; Birkelund, Svend; Christiansen, Gunna

    1994-01-01

    A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for one monoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA...... technique, the epitope was limited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The amino acid sequence homologous to the epitope is located in a linear...

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

    Science.gov (United States)

    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.

  17. Prediction of linear B-cell epitopes of hepatitis C virus for vaccine development

    Science.gov (United States)

    2015-01-01

    Background High genetic heterogeneity in the hepatitis C virus (HCV) is the major challenge of the development of an effective vaccine. Existing studies for developing HCV vaccines have mainly focused on T-cell immune response. However, identification of linear B-cell epitopes that can stimulate B-cell response is one of the major tasks of peptide-based vaccine development. Owing to the variability in B-cell epitope length, the prediction of B-cell epitopes is much more complex than that of T-cell epitopes. Furthermore, the motifs of linear B-cell epitopes in different pathogens are quite different (e. g. HCV and hepatitis B virus). To cope with this challenge, this work aims to propose an HCV-customized sequence-based prediction method to identify B-cell epitopes of HCV. Results This work establishes an experimentally verified dataset comprising the B-cell response of HCV dataset consisting of 774 linear B-cell epitopes and 774 non B-cell epitopes from the Immune Epitope Database. An interpretable rule mining system of B-cell epitopes (IRMS-BE) is proposed to select informative physicochemical properties (PCPs) and then extracts several if-then rule-based knowledge for identifying B-cell epitopes. A web server Bcell-HCV was implemented using an SVM with the 34 informative PCPs, which achieved a training accuracy of 79.7% and test accuracy of 70.7% better than the SVM-based methods for identifying B-cell epitopes of HCV and the two general-purpose methods. This work performs advanced analysis of the 34 informative properties, and the results indicate that the most effective property is the alpha-helix structure of epitopes, which influences the connection between host cells and the E2 proteins of HCV. Furthermore, 12 interpretable rules are acquired from top-five PCPs and achieve a sensitivity of 75.6% and specificity of 71.3%. Finally, a conserved promising vaccine candidate, PDREMVLYQE, is identified for inclusion in a vaccine against HCV. Conclusions This work

  18. Epitope-dependent functional effects of celiac disease autoantibodies on transglutaminase 2

    DEFF Research Database (Denmark)

    Hnida, Kathrin; Stamnaes, Jorunn; du Pré, M Fleur

    2016-01-01

    Transglutaminase 2 (TG2) is a Ca(2+)-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2...... of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients....

  19. 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

    Directory of Open Access Journals (Sweden)

    Wei Jian-Chao

    2011-07-01

    Full Text Available Abstract 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.

  20. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

  1. Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in the spike 2 domain and the nucleocapsid protein of feline infectious peritonitis virus.

    Science.gov (United States)

    Satoh, Ryoichi; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Motokawa, Kenji; Gemma, Tsuyoshi; Watanabe, Rie; Arai, Setsuo; Hohdatsu, Tsutomu

    2011-02-17

    The antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection has been recognized in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis (FIP). In the present study, we synthesized eighty-one kinds of peptides derived from the spike (S)2 domain of type I FIPV KU-2 strain, the S2 domain of type II FIPV 79-1146 strain, and the nucleocapcid (N) protein of FIPV KU-2 strain. To detect the T helper (Th)1 epitope, peripheral blood mononuclear cells (PBMCs) obtained from FIPV-infected cats were cultured with each peptide, and Th1-type immune responses were measured using feline interferon (fIFN)-γ production as an index. To detect the linear immunodominant antibody-binding epitope, we investigated the reactivity of plasma collected from FIPV-infected cats against each peptide by ELISA. Four and 2 peptides containing Th1 epitopes were identified in the heptad repeat (HR)1 and inter-helical (IH) regions of the S2 domain of type I FIPV, respectively, and these were located on the N-terminal side of the regions. In the S2 domain of type II FIPV, 2, 3, and 2 peptides containing Th1 epitopes were identified in the HR1, IH, and HR2 regions, respectively, and these were mainly located on the C-terminal side of the regions. In the S2 domain of type I FIPV, 3 and 7 peptides containing linear immunodominant antibody-binding epitopes were identified in the IH and HR2 regions, respectively. In the S2 domain of type II FIPV, 4 peptides containing linear immunodominant antibody-binding epitopes were identified in the HR2 region. The Th1 epitopes in the S2 domain of type I and II FIPV were located in different regions, but the linear immunodominant antibody-binding epitopes were mostly located in the HR2 region. Eight peptides containing Th1 epitopes were identified in N protein, and 3 peptides derived from residues 81 to 100 and 137 to 164 showed strong

  2. Analysis of potato virus Y coat protein epitopes recognized by three commercial monoclonal antibodies.

    Science.gov (United States)

    Tian, Yan-Ping; Hepojoki, Jussi; Ranki, Harri; Lankinen, Hilkka; Valkonen, Jari P T

    2014-01-01

    Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified. SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs. The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

  3. A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

    Science.gov (United States)

    Davidson, Edgar; Doranz, Benjamin J

    2014-09-01

    Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

  4. Databases and Associated Bioinformatic Tools in Studies of Food Allergens, Epitopes and Haptens – a Review

    Directory of Open Access Journals (Sweden)

    Bucholska Justyna

    2018-06-01

    Full Text Available Allergies and/or food intolerances are a growing problem of the modern world. Diffi culties associated with the correct diagnosis of food allergies result in the need to classify the factors causing allergies and allergens themselves. Therefore, internet databases and other bioinformatic tools play a special role in deepening knowledge of biologically-important compounds. Internet repositories, as a source of information on different chemical compounds, including those related to allergy and intolerance, are increasingly being used by scientists. Bioinformatic methods play a signifi cant role in biological and medical sciences, and their importance in food science is increasing. This study aimed at presenting selected databases and tools of bioinformatic analysis useful in research on food allergies, allergens (11 databases, epitopes (7 databases, and haptens (2 databases. It also presents examples of the application of computer methods in studies related to allergies.

  5. IMMUNOCAT—A Data Management System for Epitope Mapping Studies

    Directory of Open Access Journals (Sweden)

    Jo L. Chung

    2010-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  7. Some epitopes conservation in non structural 3 protein dengue virus serotype 4

    Directory of Open Access Journals (Sweden)

    Tegar A. P. Siregar

    2016-03-01

    Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify

  8. Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca2+ depletion.

    Science.gov (United States)

    Bugajska-Schretter, A; Elfman, L; Fuchs, T; Kapiotis, S; Rumpold, H; Valenta, R; Spitzauer, S

    1998-01-01

    Type I allergy to fish is a severe health problem in countries in which a large percentage of the population derive income from fishing. The aim of the study was to characterize cross-reactive IgE-binding components in six different fish species (cod, tuna, salmon, perch, carp, and eel). The effect of reducing extraction conditions, periodate treatment, and depletion of Ca2+ on binding of IgE to the allergens was investigated. Extracts were prepared under nonreducing and reducing conditions. IgE-binding components were characterized by IgE immunoblotting, and cross-reactive epitopes were studied by IgE-immunoblot inhibition experiments. To reveal calcium-sensitive or carbohydrate-containing epitopes, nitrocellulose-blotted extracts were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and periodate. Sera from all patients allergic to fish (n = 30) displayed IgE reactivity to parvalbumin, a 12 kd protein present in fish extracts from six different species. Reducing extraction conditions had no effect on IgE binding to parvalbumins, whereas periodate treatment and depletion of protein-bound calcium led to a substantial reduction of IgE binding. Parvalbumins from six different species contained cross-reactive IgE epitopes. Parvalbumin represents a cross-reactive fish allergen. It contains IgE epitopes that are sensitive to periodate treatment and Ca2+-depletion.

  9. Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility

    Directory of Open Access Journals (Sweden)

    Miguel Álvaro-Benito

    2018-05-01

    Full Text Available The major histocompatibility complex of class II (MHCII immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU] allowing the identification and label-free quantification (LFQ of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAt

  10. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

  11. Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

    DEFF Research Database (Denmark)

    Alifrangis, Michael; Christensen, Inge T; Jørgensen, Flemming S

    2004-01-01

    in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site...

  12. IgE epitopes of intact and digested Ara h 1

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Nielsen, H.; Madsen, Charlotte Bernhard

    2012-01-01

    epitopes have been suggested to be of great importance. ObjectiveThe aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. MethodsSera from five peanut allergic patients and five Brown Norway rats were used...... to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule...... to mimic epitopes using a computer-based algorithm. ResultsPatients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which...

  13. MHC class I epitope binding prediction trained on small data sets

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Nielsen, Morten; Lamberth, K.

    2004-01-01

    The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding pre...... in situations where only very limited data are available for training....

  14. Immunotherapy for Alzheimer's disease: DNA- and protein-based epitope vaccines.

    Science.gov (United States)

    Davtyan, Hayk; Petrushina, Irina; Ghochikyan, Anahit

    2014-01-01

    Active immunotherapy for Alzheimer's disease (AD) is aimed to induce antibodies specific to amyloid-beta (Aβ) that are capable to reduce the level of Aβ in the CNS of Alzheimer's disease patients. First clinical trial AN-1792 that was based on vaccination with full-length Aβ42 showed that safe and effective AD vaccine should induce high titers of anti-Aβ antibodies without activation of harmful autoreactive T cells. Replacement of self-T cell epitope with foreign epitope, keeping self-B cell epitope intact, may allow to induce high titers of anti-Aβ antibodies while avoiding the activation of T cells specific to Aβ. Here we describe the protocols for evaluation of AD DNA- or multiple antigenic peptide (MAP)-based epitope vaccines composed of Aβ(1-11) B cell epitope fused to synthetic T cell epitope PADRE (Aβ(1-11)-PADRE). All protocols could be used for testing any epitope vaccine constructed in your lab and composed of other T cell epitopes using the appropriate peptides in tests for evaluation of humoral and cellular immune responses.

  15. Expression of Hemagglutinin–Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Amir Ghaffar Shahriari

    2016-07-01

    Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

  16. A Rationally Designed TNF-α Epitope-Scaffold Immunogen Induces Sustained Antibody Response and Alleviates Collagen-Induced Arthritis in Mice.

    Directory of Open Access Journals (Sweden)

    Li Zhang

    Full Text Available The TNF-α biological inhibitors have significantly improved the clinical outcomes of many autoimmune diseases, in particular rheumatoid arthritis. However, the practical uses are limited due to high costs and the risk of anti-drug antibody responses. Attempts to develop anti-TNF-α vaccines have generated encouraging data in animal models, however, data from clinical trials have not met expectations. In present study, we designed a TNF-α epitope-scaffold immunogen DTNF7 using the transmembrane domain of diphtheria toxin, named DTT as a scaffold. Molecular dynamics simulation shows that the grafted TNF-α epitope is entirely surface-exposed and presented in a native-like conformation while the rigid helical structure of DTT is minimally perturbed, thereby rendering the immunogen highly stable. Immunization of mice with alum formulated DTNF7 induced humoral responses against native TNF-α, and the antibody titer was sustained for more than 6 months, which supports a role of the universal CD4 T cell epitopes of DTT in breaking self-immune tolerance. In a mouse model of rheumatoid arthritis, DTNF7-alum vaccination markedly delayed the onset of collagen-induced arthritis, and reduced incidence as well as clinical score. DTT is presumed safe as an epitope carrier because a catalytic inactive mutant of diphtheria toxin, CRM197 has good clinical safety records as an active vaccine component. Taken all together, we show that DTT-based epitope vaccine is a promising strategy for prevention and treatment of autoimmune diseases.

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

    Science.gov (United States)

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

    2014-12-15

    Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients. Copyright © 2014 by The American Association of Immunologists, Inc.

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

    Science.gov (United States)

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

    1998-10-15

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

  19. Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

    Science.gov (United States)

    Lu, Yudong; Li, Zhong; Teng, Huan; Xu, Hongke; Qi, Songnan; He, Jian'an; Gu, Dayong; Chen, Qijun; Ma, Hongwei

    2015-08-21

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression.

  20. Identification and fine mapping of a linear B cell epitope of human vimentin

    DEFF Research Database (Denmark)

    Dam, Catharina Essendrup; Houen, Gunnar; Hansen, Paul R.

    2014-01-01

    Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme......-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide...... sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular...

  1. CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening

    DEFF Research Database (Denmark)

    Wang, M.J.; Lamberth, K.; Harndahl, M.

    2007-01-01

    are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen......-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential....

  2. Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Breum, Solvej Østergaard; Riber, Ulla

    2014-01-01

    Background: Major histocompatibility complex (MHC) class I peptide binding and presentation are essential for antigen-specific activation of cytotoxic T lymphocytes (CTLs) and swine MHC class I molecules, also termed swine leukocyte antigens (SLA), thus play a crucial role in the process that leads...... to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. Findings: Four SwIV derived peptides were...

  3. Structural analysis of linear and conformational epitopes of allergens

    Science.gov (United States)

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

    2009-01-01

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

  4. Novel approaches to cancer targeting using epitope-binding properties that mimic monoclonal antibodies

    International Nuclear Information System (INIS)

    1998-01-01

    The investigators have extensive experience in all of the techniques required for this project including: animal models of tumors, quantitative autoradiography, radiochemistry, peptide synthesis, organic synthesis, molecular biology, kinetic modeling and radionuclide imaging both with single photon and PET agents. Preliminary results and progress in the following areas are presented: (1) Establishment of an in vivo tumor model and successful targeting of this tumor using monoclonal antibodies raised to p185 erbB2 , (2a) Screening of a synthetic peptide combinatorial library, (2b) Screening of a phage display peptide library, (3) Determination of the epitope recognized by ICR12, (4) Radiolabeling with 99m Tc, 18 F and * I (radioiodines), (5) High resolution positron emission tomography (PET) studies of tumors, and (6) Development of a high resolution (∼mm) PET camera

  5. Impact of epitope specificity and precursor maturation in pro-B-type natriuretic peptide measurement

    DEFF Research Database (Denmark)

    Goetze, J.P.; Dahlstrom, U.; Alehagen, U.

    2008-01-01

    with different epitope specificities in a cohort of elderly patients presenting with symptoms associated with heart failure (n = 415). RESULTS: Comparison of N-terminal proBNP with proBNP 1-76 measurement in plasma revealed a high correlation on regression analysis (r(2) = 0.91, P ..., the proBNP 1-76 assay measured lower concentrations in the high range than the N-terminal proBNP assay. Correlations between assay measurements in a clinical setting were comparable for all the assays (r(2) approximately 0.57-0.83), and ROC analyses revealed area-under-the-curve values ranging between 0......BACKGROUND: Cardiac-derived natriuretic peptides are sensitive plasma markers of cardiac dysfunction. Recent reports have disclosed a more complex molecular heterogeneity of B-type natriuretic peptide precursor (proBNP)-derived peptides than previously suggested. In this study, we examined...

  6. 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

    International Nuclear Information System (INIS)

    Yu Hua; Jiang Lifang; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-01-01

    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

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

    DEFF Research Database (Denmark)

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

    2017-01-01

    -reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification...

  8. Carrier protein influences immunodominance of a known epitope: implication in peptide vaccine design.

    Science.gov (United States)

    Ghosh, Moumita; Solanki, Ashish K; Roy, Koushik; Dhoke, Reema R; Ashish; Roy, Syamal

    2013-09-23

    We investigated how the processing of a given antigen by antigen presenting cells (APC) is dictated by the conformation of the antigen and how this governs the immunodominance hierarchy. To address the question, a known immunodominant sequence of bacteriophage lambda repressor N-terminal sequence 12-26 [λR(12-26)] was engineered at the N and C termini of a heterologous leishmanial protein, Kinetoplastid membrane protein-11 (KMP-11); the resulting proteins were defined as N-KMP-11 and C-KMP-11 respectively. The presence of λR(12-26) in N-KMP-11 and C-KMP-11 was established by western blot analysis with antibody to λR(12-26) peptide. N-KMP-11 but not C-KMP-11 could stimulate the anti λR(12-26) T-cell clonal population very efficiently in the presence of APCs. Priming of BALB/c mice with N-KMP-11 or C-KMP-11 generated similar levels of anti-KMP-11 IgG, but anti-λR(12-26) specific IgG was observed only upon priming with N-KMP-11. Interestingly, uptake of both N-KMP-11 and C-KMP-11 by APCs was similar but catabolism of N-KMP-11 but not C-KMP-11 was biphasic and fast at the initial time point. Kratky plots of small angle X-ray scattering showed that while N-KMP-11 adopts flexible Gaussian type of topology, C-KMP-11 prefers Globular nature. To show that KMP-11 is not unique as a carrier protein, an epitope (SPITBTNLBTMBK) of Plasmodium yoelii (PY) apical membrane protein 1[AMA-1 (136-148)], is placed at the C and N terminals of a dominant T-cell epitope of ovalbumin protein OVA(323-339) and the resulting peptides are defined as PY-OVA and OVA-PY respectively. Interestingly, only OVA-PY could stimulate anti-OVA T-cells and produce IgG response upon priming of BALB/c mice with it. Thus for rational design of peptide vaccine it is important to place the dominant epitope appropriately in the context of the carrier protein. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-12-04

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

  10. Mapping of epitopes on Poa p I and Lol p I allergens with monoclonal antibodies.

    Science.gov (United States)

    Lin, Z W; Ekramoddoullah, A K; Jaggi, K S; Dzuba-Fischer, J; Rector, E; Kisil, F T

    1990-01-01

    Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

    Science.gov (United States)

    Lu, Zhaohua; Perkins, Hillary M.

    2014-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. PMID:24351753

  12. HLA-A2–Restricted Cytotoxic T Lymphocyte Epitopes from Human Heparanase as Novel Targets for Broad-Spectrum Tumor Immunotherapy

    Directory of Open Access Journals (Sweden)

    Ting Chen

    2008-09-01

    Full Text Available Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2– restricted cytotoxic T lymphocyte (CTL epitopes in the protein of human Hpa. For this purpose, HLA-A2–restricted CTL epitopes were identified using the following four-step procedure: 1 a computer-based epitope prediction from the amino acid sequence of human Hpa, 2 a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3 stimulation of the primary T-cell response against the predicted peptides in vitro, and 4 testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525, 277-285 (KMLKSFLKA, Hpa277, and 405-413 (WLSLLFKKL, Hpa405 could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ–producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2–restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide–based vaccines may be useful for the immunotherapy for patients with advanced tumors.

  13. Epitope characterization of the ADA response directed against a targeted immunocytokine.

    Science.gov (United States)

    Stubenrauch, Kay; Künzel, Christian; Vogel, Rudolf; Tuerck, Dietrich; Schick, Eginhard; Heinrich, Julia

    2015-10-10

    Targeted immunocytokines (TICs) display potent activity in selective tumor suppression. This class of multi domain biotherapeutics (MDBs) is composed of the three major domains Fab, Fc, and a cytokine which may induce a complex polyclonal anti-drug antibody (ADA) response. However, classical ADA assays usually are not suitable to specify ADAs and to identify the immunogenic domains of a TIC. The purpose of the present study was to establish epitope characterization of ADA responses in order to specify immunogenic responses against a TIC and their direct impact on the pharmacokinetic profile, safety, and efficacy. Based on standard ADA screening and confirmation assays, respectively, domain detection assays (DDAs) and domain competition assays (DCAs) were established and compared by the use of 12 ADA-positive samples obtained from a cynomolgus monkey study in early development. Both domain-specific assays were sensitive enough to preserve the positive screening assay result and revealed an overall accordance for the evaluation of domain-specific ADA responses. About half of the samples displayed one ADA specificity, either for the Fab or for the cytokine (Cy) domain, and the remaining samples showed a combination of Fab-specific and Cy-specific ADA fractions. Fc-specific ADAs occurred in only one sample. In-depth comparison of DCAs and DDAs showed that both assays appeared to be appropriate to assess multi-specific ADA responses as well as minor ADA fractions. An advantage of DCAs is typically a fast and easy assay establishment, whereas, DDAs in some cases may be superior to assess low abundant ADAs in multi-specific responses. Our results reveal that both approaches benefit from thorough reagent development as an essential precondition for reliable epitope characterization of ADA responses. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Epitope selection from an uncensored peptide library displayed on avian leukosis virus

    International Nuclear Information System (INIS)

    Khare, Pranay D.; Rosales, Ana G.; Bailey, Kent R.; Russell, Stephen J.; Federspiel, Mark J.

    2003-01-01

    Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10 6 different members was generated from a plasmid library of approximately 5 x 10 6 diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries

  15. Systematic screening for novel, serologically reactive Hepatitis E Virus epitopes

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    Osterman Andreas

    2012-01-01

    Full Text Available Abstract Background The National Institutes of Health classified Hepatitis E as an emerging disease since Hepatitis E Virus (HEV is the major cause of acute hepatitis in developing countries. Interestingly, an increasing number of sporadic cases of HEV infections are described in industrialized countries as zoonosis from domestic livestock. Despite the increasing relevance of this pathogen in clinical virology, commercial antibody assays are mainly based on fragments of HEV open reading frame (ORF 2 and ORF3. The largest ORF1 (poly-protein, however, is not part of current testing formats. Methods From a synthesized full length HEV genotype 1 cDNA-bank we constructed a complete HEV gene library consisting of 15 respective HEV ORF domains. After bacterial expression and purification of nine recombinant HEV proteins under denaturating conditions serum profiling experiments using 55 sera from patients with known infection status were performed in microarray format. SPSS software assessed the antigenic potential of these nine ORF domains in comparison to seven commercial HEV antigens (genotype 1 and 3 by performing receiver operator characteristics, logistic regression and correlation analysis. Results HEV antigens produced with our method for serum profiling experiments exhibit the same quality and characteristics as commercial antigens. Serum profiling experiments detected Y, V and X domains as ORF1-antigens with potentially comparable diagnostic significance as the well established epitopes of ORF2 and ORF3. However no obvious additional increase in sensitivity or specificity was achieved in diagnostic testing as revealed by bioinformatic analysis. Additionally we found that the C-terminal domain of the potential transmembrane protein ORF3 is responsible for IgG and IgM seroreactivity. Data suggest that there might be a genotype specific seroreactivity of homologous ORF2-antigens. Conclusions The diagnostic value of identified ORF1 epitopes might

  16. PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.

    Directory of Open Access Journals (Sweden)

    Wenhan Chen

    Full Text Available 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/

  17. Identification and characterization of two linear epitope motifs in hepatitis E virus ORF2 protein.

    Directory of Open Access Journals (Sweden)

    Heng Wang

    Full Text Available Hepatitis E virus (HEV is responsible for hepatitis E, which represents a global public health problem. HEV genotypes 3 and 4 are reported to be zoonotic, and animals are monitored for HEV infection in the interests of public hygiene and food safety. The development of novel diagnostic methods and vaccines for HEV in humans is thus important topics of research. Opening reading frame (ORF 2 of HEV includes both linear and conformational epitopes and is regarded as the primary candidate for vaccines and diagnostic tests. We investigated the precise location of the HEV epitopes in the ORF2 protein. We prepared four monoclonal antibodies (mAbs against genotype 4 ORF2 protein and identified two linear epitopes, G438IVIPHD444 and Y457DNQH461, corresponding to two of these mAbs using phage display biopanning technology. Both these epitopes were speculated to be universal to genotypes 1, 2, 3, 4, and avian HEVs. We also used two 12-mer fragments of ORF2 protein including these two epitopes to develop a peptide-based enzyme-linked immunosorbent assay (ELISA to detect HEV in serum. This assay demonstrated good specificity but low sensitivity compared with the commercial method, indicating that these two epitopes could serve as potential candidate targets for diagnosis. Overall, these results further our understanding of the epitope distribution of HEV ORF2, and provide important information for the development of peptide-based immunodiagnostic tests to detect HEV in serum.

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

    Science.gov (United States)

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

    2017-09-15

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

  19. Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): preparation and immunological analysis of vaccine efficacy.

    Science.gov (United States)

    Masuko, Kazutaka; Wakita, Daiko; Togashi, Yuji; Kita, Toshiyuki; Kitamura, Hidemitsu; Nishimura, Takashi

    2015-01-01

    To elucidate the immunologic mechanisms of artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which indicated a great vaccine efficacy in human cancers, we prepared ovalbumin (OVA)-H/K-HELP by conjugating killer and helper epitopes of OVA-model tumor antigen via a glycine-linker. Vaccination of C57BL/6 mice with OVA-H/K-HELP (30 amino acids) but not with short peptides mixture of class I-binding peptide (8 amino-acids) and class II-binding peptide (17 amino-acids) combined with adjuvant CpG-ODN (cytosine-phosphorothioate-guanine oligodeoxynucleotides), induced higher numbers of OVA-tetramer-positive CTL with concomitant activation of IFN-γ-producing CD4(+) Th1 cells. However, replacement of glycine-linker of OVA-H/K-HELP with other peptide-linker caused a significant decrease of vaccine efficacy of OVA-H/K-HELP. In combination with adjuvant CpG-ODN, OVA-H/KHELP exhibited greater vaccine efficacy compared with short peptides vaccine, in both preventive and therapeutic vaccine models against OVA-expressing EG-7 tumor. The elevated vaccine efficacy of OVAH/K-HELP might be derived from the following mechanisms: (i) selective presentation by only professional dendritic cells (DC) in vaccinated draining lymph node (dLN); (ii) a long-term sustained antigen presentation exerted by DC to stimulate both CTL and Th1 cells; (iii) formation of three cells interaction among DC, Th and CTL. In comparative study, H/K-HELP indicated stronger therapeutic vaccine efficacy compared with that of extended class I synthetic long peptide, indicating that both the length of peptide and the presence of Th epitope peptide were crucial aspects for preparing artificially synthesized H/K-HELP vaccine. Copyright © 2014 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  20. Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks

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    Chenxi Li

    2016-11-01

    Full Text Available 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.

  1. Epitopes associated with MHC restriction site of T cells. III. I-J epitope on MHC-restricted T helper cells

    International Nuclear Information System (INIS)

    Asano, Y.; Nakayama, T.; Kubo, M.; Yagi, J.; Tada, T.

    1987-01-01

    I-J epitopes were found to be associated with the functional site of the class II MHC-restricted helper T (Th) cells: Virtually all of the H-2k-restricted Th cell function of H-2kxbF1 T cells was inhibited by the anti-I-Jk mAb, leaving the H-2b-restricted function unaffected. The I-Jk epitope was inducible in Th cells of different genotype origin according to the environmental class II antigens present in the early ontogeny of T cells. Although above results suggested that I-J is the structure reflecting the inducible MHC restriction specificity, further studies revealed some interesting controversies: First, the I-J phenotype did not always correlate with the class II restriction specificity, e.g., I-Ab-restricted Th from 5R was I-Jk-positive, whereas I-Ak-restricted Th of 4R was not. Second, there was no trans expression of parental I-J phenotypes and restriction specificities in F1 Th, e.g., the I-J phenotype was detected only on I-Ab-restricted Th of (4R X 5R)F1, whereas it was absent on I-Ak-restricted Th. This strict linkage between the restriction specificity and I-J phenotype was also found on Th cells developed in bone marrow chimera constructed with intra-H-2-recombinant mice. The expression of I-Jk was always associated with the restriction specificity of the relevant host. Thus, the restriction specificity of Th cells followed the host type, and the I-J expression on Th was exactly the same as that expressed by the host haplotype. These results indicate that I-J is an isomorphic structure adaptively expressed on Th cells that is involved in the unidirectional regulatory cell interactions, and that the polymorphism cannot be explained merely by the restriction specificity of the conventional T cell receptor heterodimer

  2. Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K.

    2007-01-01

    BACKGROUND: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein....... of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL.All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php....

  3. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

    DEFF Research Database (Denmark)

    Bresciani, Anne Gøther; Paul, Sinu; Schommer, Nina

    2016-01-01

    or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially...

  4. A novel monoclonal antibody to a defined peptide epitope in MUC16

    DEFF Research Database (Denmark)

    Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa

    2015-01-01

    with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent...... immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry...

  5. Reliable B cell epitope predictions: impacts of method development and improved benchmarking

    DEFF Research Database (Denmark)

    Kringelum, Jens Vindahl; Lundegaard, Claus; Lund, Ole

    2012-01-01

    biomedical applications such as; rational vaccine design, development of disease diagnostics and immunotherapeutics. However, experimental mapping of epitopes is resource intensive making in silico methods an appealing complementary approach. To date, the reported performance of methods for in silico mapping...... evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version...

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

    Science.gov (United States)

    Chen, Xueni; Dreskin, Stephen C

    2017-06-01

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

  7. Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

    Directory of Open Access Journals (Sweden)

    Piontkivska Helen

    2009-07-01

    Full Text Available Abstract Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1 regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007, we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms. The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges

  8. Mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats.

    Science.gov (United States)

    Takano, Tomomi; Tomiyama, Yoshika; Katoh, Yasuichiroh; Nakamura, Michiyo; Satoh, Ryoichi; Hohdatsu, Tsutomu

    2011-03-01

    We previously prepared neutralizing monoclonal antibody (MAb)-resistant (mar) mutant viruses using a laboratory strain feline infectious peritonitis virus (FIPV) 79-1146 (Kida et al., 1999). Mar mutant viruses are mutated several amino acids of the neutralizing epitope of Spike protein, compared with the parent strain, FIPV 79-1146. We clarified that MAb used to prepare mar mutant viruses also lost its activity to enhance homologous mar mutant viruses, strongly suggesting that neutralizing and antibody-dependent enhancing epitopes are present in the same region in the strain FIPV 79-1146. We also discovered that amino acid mutation in the neutralizing epitope reduced viral replication in monocytes/macrophages. We also demonstrated that the mutation or deletion of two nucleotides in 7b gene abrogate the virulence of strain FIPV 79-1146. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

    Directory of Open Access Journals (Sweden)

    Ajda Biček

    2004-01-01

    Full Text Available Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of turkey, pheasant and peafowl. Chicken IgY light chain specific mAb 3E10 revealed the presence of common epitopes on immunoglobulins of turkey, pheasant and sparrow. Monoclonal antibody clone 1F5/3G2 was used to prepare horseradish peroxidase (HRP conjugate and immunoadsorbent column. Conjugated mAbs were demonstrated to be excellent secondary antibodies for diagnostics of certain infections in different avian species. Since they do not react with mammalian immunoglobulins using our mAbs as secondary antibodies in human serodiagnostics would minimize background staining that appears when using mouse detection system. In dot immunobinding assay (DIBA and immunoblot assay they recognized specific IgY antibodies against Mycoplasma synoviae, Mycoplasma gallisepticum and Newcastle disease virus in sera of infected or vaccinated birds. Immunoadsorption as a method for removal of IgY from samples in which Mycoplasma synoviae specific IgY was predominant immunoglobulin class enabled more exact demonstration of specific IgA and IgM antibodies. Herein we are presenting effective mAbs useful in diagnostics of avian and mammalian infections as well as in final steps of detection and purification of chicken antibodies and their subunits produced in vivo or in vitro as polyclonal, monoclonal or recombinant antibodies.

  10. Analysis of Conformational B-Cell Epitopes in the Antibody-Antigen Complex Using the Depth Function and the Convex Hull.

    Directory of Open Access Journals (Sweden)

    Wei Zheng

    Full Text Available 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.

  11. Identification of murine T-cell epitopes in Ebola virus nucleoprotein

    International Nuclear Information System (INIS)

    Simmons, Graham; Lee, Anee; Rennekamp, Andrew J.; Fan Xin; Bates, Paul; Shen Hao

    2004-01-01

    CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2 d -restricted epitope (NP279-288) and two H-2 b -restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection

  12. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

    DEFF Research Database (Denmark)

    Buus, Søren; Rockberg, Johan; Forsström, Björn

    2012-01-01

    Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning...... against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high......-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against...

  13. A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies

    DEFF Research Database (Denmark)

    Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah

    2017-01-01

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

  14. Structural and Dynamic Insight into Hirudin Epitopes-HLA- DRB1 ...

    African Journals Online (AJOL)

    It is produced in the salivary glands of the leech Hirudo .... for epitope selection has a specific algorithm and was developed in ..... from Plasmodium falciparum pre-erythrocytic-stage antigens ... Comar M. Identification of a vaccine against.

  15. Antibodies against linear epitopes on Goodpasture autoantigen in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis.

    Science.gov (United States)

    Jia, Xiao-Yu; Yu, Jun-Tao; Hu, Shui-Yi; Li, Jian-Nan; Wang, Miao; Wang, Chen; Chen, Min; Cui, Zhao; Zhao, Ming-Hui

    2017-09-01

    In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr ≤ 133 μmol/L), 70% of patients with moderate renal dysfunction (133 μmol/L  600 μmol/L) (P = 0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of E A (P4) and E B (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r = 0.764, P = 0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P = 0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.

  16. Substantial gaps in knowledge of Bordetella pertussis antibody and T cell epitopes relevant for natural immunity and vaccine efficacy

    Science.gov (United States)

    Vaughan, Kerrie; Seymour, Emily; Peters, Bjoern; Sette, Alessandro

    2016-01-01

    The recent increase in whooping cough in vaccinated populations has been attributed to waning immunity associated with the acellular vaccine. The Immune Epitope Database (IEDB) is a repository of immune epitope data from the published literature and includes T cell and antibody epitopes for human pathogens. The IEDB conducted a review of the epitope literature, which revealed 300 Bordetella pertussis-related epitopes from 39 references. Epitope data are currently available for six virulence factors of B. pertussis: pertussis toxin, pertactin, fimbrial 2, fimbrial 3, adenylate cyclase and filamentous hemagglutinin. The majority of epitopes were defined for antibody reactivity; fewer T cell determinants were reported. Analysis of available protective correlates data revealed a number of candidate epitopes; however few are defined in humans and few have been shown to be protective. Moreover, there are a limited number of studies defining epitopes from natural infection versus whole cell or acellular/subunit vaccines. The relationship between epitope location and structural features, as well as antigenic drift (SNP analysis) was also investigated. We conclude that the cumulative data is yet insufficient to address many fundamental questions related to vaccine failure and this underscores the need for further investigation of B. pertussis immunity at the molecular level. PMID:24530743

  17. Microarray glycan profiling reveals algal fucoidan epitopes in diverse marine metazoans

    DEFF Research Database (Denmark)

    Asunción Salmeán, Armando; Hervé, Cécile; Jørgensen, Bodil

    2017-01-01

    Despite the biological importance and pharmacological potential of glycans from marine organisms, there are many unanswered questions regarding their distribution, function, and evolution. Here we describe microarray-based glycan profiling of a diverse selection of marine animals using antibodies...... raised against fucoidan isolated from a brown alga. We demonstrate the presence of two fucoidan epitopes in six animals belonging to three phyla including Porifera, Molusca, and Chordata. We studied the spatial distribution of these epitopes in Cliona celata ("boring sponge") and identified...

  18. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    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.

  19. Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Conformational occlusion of blockade antibody epitopes, a novel mechanism of GII.4 human norovirus immune evasion

    OpenAIRE

    Lindesmith, Lisa C.; Mallory, Michael L.; Debbink, Kari; Donaldson, Eric F.; Brewer-Jensen, Paul D.; Swann, Excel W.; Sheahan, Timothy P.; Graham, Rachel L.; Beltramello, Martina; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S.

    2018-01-01

    ABSTRACT Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach f...

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

    OpenAIRE

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

    2013-01-01

    Background The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Findings Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. T...

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

    Science.gov (United States)

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

    2013-01-01

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

  3. Multiple epitopes in a dodecapeptide of myelin basic protein determined bymonoclonal antibodies

    International Nuclear Information System (INIS)

    Price, J.O.; Whitaker, J.N.; Vasu, R.I.; Metzger, D.W.

    1986-01-01

    Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89

  4. Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Luciana Machado Bastos

    2016-06-01

    Full Text Available Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2 is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN, as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b, mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

  5. Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope

    International Nuclear Information System (INIS)

    Yao, Shugang; Qi, Jianxun; Liu, Jun; Chen, Rong; Pan, Xiaocheng; Li, Xiaoying; Gao, Feng; Xia, Chun

    2011-01-01

    The equine MHC class I molecule was crystallized in complex with β 2 -microglobulin and a CTL epitope and X-ray diffraction data were collected to 2.3 Å resolution. In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse β 2 -microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195–206) was refolded and crystallized. The crystal, which belonged to space group P2 1 , diffracted to 2.3 Å resolution and had unit-cell parameters a = 82.5, b = 71.4, c = 99.8 Å, β = 102.9°. The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope

  6. Structural Simulation of MHC-peptide Interactions using T-cell Epitope in Iron-acquisition Protein of N. meningitides for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Namrata Mishra

    2010-12-01

    Full Text Available The present work uses a structural simulation approach to identify the potential target vaccine candidates or T cell epitopes (antigenic region that can activate T cell response in two iron acquisition proteins from Neisseria. An iron regulated outer membrane protein frpB: extracellular, [NMB1988], and a Major ferric Iron-binding protein fbpA: periplasmic, [NMB0634] critical for the survival of the pathogen in the host were used. Ten novel promiscuous epitopes from the two iron acquisition proteins were identified using bioinformatics interface. Of these epitopes, 630VQKAVGSIL638 present on frpB with high binding affinity for allele HLA*DR1 was identified with an anchor position at P2, an aliphatic residue at P4 and glycine at P6 making it thereby a potential quality choice for linking peptide-loaded MHC dynamics to T-cell activation and vaccine constructs. The feasibility and structural binding of predicted peptide to the respective HLA allele was investigated by molecular modeling and template-based structural simulation. The conformational properties of the linear peptide were investigated by molecular dynamics using GROMOS96 package and Swiss PDB viewer.

  7. Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

    Directory of Open Access Journals (Sweden)

    Paula S Santos

    Full Text Available Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

  8. Overlapping CD8+ and CD4+ T-cell epitopes identification for the progression of epitope-based peptide vaccine from nucleocapsid and glycoprotein of emerging Rift Valley fever virus using immunoinformatics approach.

    Science.gov (United States)

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-12-01

    Rift Valley fever virus (RVFV) is an emergent arthropod-borne zoonotic infectious viral pathogen which causes fatal diseases in the humans and ruminants. Currently, no effective and licensed vaccine is available for the prevention of RVFV infection in endemic as well as in non-endemic regions. So, an immunoinformatics-driven genome-wide screening approach was performed for the identification of overlapping CD8+ and CD4+ T-cell epitopes and also linear B-cell epitopes from the conserved sequences of the nucleocapsid (N) and glycoprotein (G) of RVFV. We identified overlapping 99.39% conserved 1 CD8+ T-cell epitope (MMHPSFAGM) from N protein and 100% conserved 7 epitopes (AVFALAPVV, LAVFALAPV, FALAPVVFA, VFALAPVVF, IAMTVLPAL, FFDWFSGLM, and FLLIYLGRT) from G protein and also identified IL-4 and IFN-γ induced (99.39% conserved) 1 N protein CD4+ T-cell epitope (HMMHPSFAGMVDPSL) and 100% conserved 5 G protein CD4+ T-cell epitopes (LPALAVFALAPVVFA, PALAVFALAPVVFAE, GIAMTVLPALAVFAL, GSWNFFDWFSGLMSW, and FFLLIYLGRTGLSKM). The overlapping CD8+ and CD4+ T-cell epitopes were bound with most conserved HLA-C*12:03 and HLA-DRB1*01:01, respectively with the high binding affinity (kcal/mol). The combined population coverage analysis revealed that the allele frequencies of these epitopes are high in endemic and non-endemic regions. Besides, we found 100% conserved and non-allergenic 2 decamer B-cell epitopes, GVCEVGVQAL and RVFNCIDWVH of G protein had the sequence similarity with the nonamer CD8+ T-cell epitopes, VCEVGVQAL and RVFNCIDWV, respectively. Consequently, these epitopes may be used for the development of epitope-based peptide vaccine against emerging RVFV. However, in vivo and in vitro experiments are required for their efficient use as a vaccine. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. B-1 cell immunoglobulin directed against oxidation-specific epitopes

    Directory of Open Access Journals (Sweden)

    Dimitrios eTsiantoulas

    2013-01-01

    Full Text Available Natural antibodies (NAbs are pre-existing antibodies with germline origin that arise in the absence of previous exposure to foreign antigens. NAbs are produced by B-1 lymphocytes and are primarily of the IgM isotype. There is accumulating evidence that - in addition to their role in antimicrobial host defense - NAbs exhibit important housekeeping functions by facilitating the non-immunogenic clearance of apoptotic cells as well as the removal of (neo-self antigens. These properties are largely mediated by the ability of NAbs to recognize highly conserved and endogenously generated structures, which are exemplified by so-called oxidation-specific epitopes (OSEs that are products of lipid peroxidation. The generation of OSEs as well as their interaction with the immune system have been studied extensively in the context of atherosclerosis, a chronic inflammatory disease of the vascular wall that is characterized by the accumulation of cellular debris and oxidized low-density lipoproteins (OxLDL. Both apoptotic cells as well as OxLDL carry OSEs that are targeted by NAbs. Therefore, OSEs represent stress-induced neo-self structures that mediate recognition of metabolic waste (e.g. cellular debris by NAbs, allowing its safe disposal, which has fundamental implications in health and disease.

  10. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

    Science.gov (United States)

    Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

  11. A comparative in silico linear B-cell epitope prediction and characterization for South American and African Trypanosoma vivax strains.

    Science.gov (United States)

    Guedes, Rafael Lucas Muniz; Rodrigues, Carla Monadeli Filgueira; Coatnoan, Nicolas; Cosson, Alain; Cadioli, Fabiano Antonio; Garcia, Herakles Antonio; Gerber, Alexandra Lehmkuhl; Machado, Rosangela Zacarias; Minoprio, Paola Marcella Camargo; Teixeira, Marta Maria Geraldes; de Vasconcelos, Ana Tereza Ribeiro

    2018-02-27

    Trypanosoma vivax is a parasite widespread across Africa and South America. Immunological methods using recombinant antigens have been developed aiming at specific and sensitive detection of infections caused by T. vivax. Here, we sequenced for the first time the transcriptome of a virulent T. vivax strain (Lins), isolated from an outbreak of severe disease in South America (Brazil) and performed a computational integrated analysis of genome, transcriptome and in silico predictions to identify and characterize putative linear B-cell epitopes from African and South American T. vivax. A total of 2278, 3936 and 4062 linear B-cell epitopes were respectively characterized for the transcriptomes of T. vivax LIEM-176 (Venezuela), T. vivax IL1392 (Nigeria) and T. vivax Lins (Brazil) and 4684 for the genome of T. vivax Y486 (Nigeria). The results presented are a valuable theoretical source that may pave the way for highly sensitive and specific diagnostic tools. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Molecular cloning, expression, IgE binding activities and in silico epitope prediction of Per a 9 allergens of the American cockroach

    Science.gov (United States)

    Yang, Haiwei; Chen, Hao; Jin, Min; Xie, Hua; He, Shaoheng; Wei, Ji-Fu

    2016-01-01

    Per a 9 is a major allergen of the American cockroach (CR), which has been recognized as an important cause of imunoglobulin E-mediated type I hypersensitivity worldwide. However, it is not neasy to obtain a substantial quantity of this allergen for use in functional studies. In the present study, the Per a 9 gene was cloned and expressed in Escherichia coli (E. coli) systems. It was found that 13/16 (81.3%) of the sera from patients with allergies caused by the American CR reacted to Per a 9, as assessed by enzyme-linked immunosorbent assay, confirming that Per a 9 is a major allergen of CR. The induction of the expression of CD63 and CCR3 in passively sensitized basophils (from sera of patients with allergies caused by the American CR) by approximately 4.2-fold indicated that recombinant Per a 9 was functionally active. Three immunoinformatics tools, including the DNASTAR Protean system, Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the potential B cell epitopes, while Net-MHCIIpan-2.0 and NetMHCII-2.2 were used to predict the T cell epitopes of Per a 9. As a result, we predicted 11 peptides (23–28, 39–46, 58–64, 91–118, 131–136, 145–154, 159–165, 176–183, 290–299, 309–320 and 338–344) as potential B cell linear epitopes. In T cell prediction, the Per a 9 allergen was predicted to have 5 potential T cell epitope sequences, 119–127, 194–202, 210–218, 239–250 and 279–290. The findings of our study may prove to be useful in the development of peptide-based vaccines to combat CR-induced allergies. PMID:27840974

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Charles D. Morris

    2017-02-01

    Full Text Available 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.

  15. Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis-Infected Cattle

    Science.gov (United States)

    Goff, Will L.; McElwain, Terry F.; Suarez, Carlos E.; Johnson, Wendell C.; Brown, Wendy C.; Norimine, Junzo; Knowles, Donald P.

    2003-01-01

    The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool. PMID:12522037

  16. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    2010-07-01

    Full Text Available Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration

  17. A Novel Multi-Epitope Vaccine For Cross Protection Against Hepatitis C Virus (HCV: An Immunoinformatics Approach

    Directory of Open Access Journals (Sweden)

    Mokhtar Nosrati

    2017-02-01

    Full Text Available Background: Hepatitis C virus (HCV causes acute and chronic human hepatitis infections. Due to the high genetic diversity and high rates of mutations in the genetic material so far there is no approved vaccine against HCV. Materials and Methods: The aim of this study was to determination B and T cell conserved epitopes of E1 and E2 proteins from HCV and construction of a chimeric peptide as a novel epitope based vaccine for cross-protection against the virus. For this, one B and T-cell epitope from both E1 and E2 which was predicted by EPMLR and Propred-1 server and had the highest score and antigenicity in VaxiJen 2.0 and PAP servers were selected for construction of chimeric protein as a multi-epitope vaccine. Results: The results of this study showed that the chimeric peptide had high antigenicity score and stability.Results also showed that most epitopes of E1 were located in two spectra consist of (45-65,88-107 and 148-182 while the results about B-cell epitopes of E2 showed that this protein had much less epitope than E1. The most epitope predicted for E2 were located in (12-24 and 35-54 spectra Conclusion:  In conclusion, epitope based vaccine which was designed by immunoinformatics methods could be considered as a novel and effective vaccine for cross-protection against HCV infection.

  18. Can mutational GC-pressure create new linear B-cell epitopes in herpes simplex virus type 1 glycoprotein B?

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich

    2009-01-01

    We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ).

  19. HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor

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    Tsukahara Tomohide

    2009-06-01

    Full Text Available Abstract Background To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2. Methods Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201+ patients with osteosarcoma using limiting dilution (LD/mixed lymphocyte peptide culture (MLPC followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by 51Cr release assay. Results Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2 × 10-7 to 5 × 10-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2. Conclusion These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2011-05-01

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

  2. Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors

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    Moustafa Hamze

    2017-05-01

    Full Text Available The chimeric antibodies anti-CD20 rituximab (Rtx and anti-TNFα infliximab (Ifx induce antidrug antibodies (ADAs in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.

  3. Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

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    Bifang He

    2013-01-01

    Full Text Available Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30, I37, D45, E84, V88, EPMGTANIQLH (92–102, VPP (131–133, Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

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

    Science.gov (United States)

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

    2017-12-01

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

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

    Science.gov (United States)

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

    2013-04-30

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

  6. Exploratory study on Th1 epitope-induced protective immunity against Coxiella burnetii infection.

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    Xiaolu Xiong

    Full Text Available Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-A(b based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4(+ T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4(+ T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4(+ T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.

  7. Genotypic and epitope characteristics of group A porcine rotavirus strains circulating in Canada.

    Science.gov (United States)

    Naseer, Omer; Jarvis, Matthew C; Ciarlet, Max; Marthaler, Douglas G

    2017-07-01

    Surveillance of Rotavirus A (RVA) infections in North America swine populations are limited and not performed over a significant time period to properly assess the diversity of RVA strains in swine. The VP7 (G) and VP4 (P) genes of 32 Canadian RVA strains, circulating between 2009 and 2015 were sequenced, identifying the G3P[13], G5P[7], G9P[7], G9[13], and G9[19] genotype combinations. The Canadian RVA strains were compared to the RVA strains present in the swine ProSystems RCE rotavirus vaccine. The comparison revealed multiple amino acid differences in the G and P antigenic epitopes, regardless of the G and P genotypes but specifically in the Canadian G3, P[13] and P[19] genotypes. Our study further contributes to the characterization of RVA's evolution and disease mitigation among swine, which may optimize target vaccine design, thereby minimizing RVA disease in this economically important animal population. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

    Science.gov (United States)

    Abbott, Jeffrey R; Palmer, Guy H; Howard, Chris J; Hope, Jayne C; Brown, Wendy C

    2004-12-01

    Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

  9. The use of HPLC-MS in T-cell epitope identification.

    Science.gov (United States)

    Lemmel, Claudia; Stevanović, Stefan

    2003-03-01

    The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).

  10. Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen

    DEFF Research Database (Denmark)

    Birkelund, Svend; Mygind, P; Holm, A

    1996-01-01

    this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding......Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against...... with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized...

  11. Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

    2018-05-03

    Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

  12. In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

    Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

  13. Mature Epitope Density - A strategy for target selection based on immunoinformatics and exported prokaryotic proteins

    DEFF Research Database (Denmark)

    Santos, Anderson R; Pereira, Vanessa Bastos; Barbosa, Eudes

    2013-01-01

    . However, currently available tools do not account for the concentration of epitope products in the mature protein product and its relation to the reliability of target selection. RESULTS: We developed a computational strategy based on measuring the epitope's concentration in the mature protein, called...... Mature Epitope Density (MED). Our method, though simple, is capable of identifying promising vaccine targets. Our online software implementation provides a computationally light and reliable analysis of bacterial exoproteins and their potential for vaccines or diagnosis projects against pathogenic...... proteins were confirmed as related. There was no experimental evidence of antigenic or pathogenic contributions for three of the highest MED-scored Mtb proteins. Hence, these three proteins could represent novel putative vaccine and drug targets for Mtb. A web version of MED is publicly available online...

  14. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano

    2010-01-01

    antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...... of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O......-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr(77)), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin...

  15. Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

    Science.gov (United States)

    Brown, W C; Zhao, S; Woods, V M; Tripp, C A; Tetzlaff, C L; Heussler, V T; Dobbelaere, D A; Rice-Ficht, A C

    1993-01-01

    Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1

  16. Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis.

    Science.gov (United States)

    Brito, J A; Preston, J F; Dickson, D W; Giblin-Davis, R M; Williams, D S; Aldrich, H C; Rice, J D

    2003-09-01

    The synthesis and localization of an endospore surface epitope associated with the development of Pasteuria penetrans was determined using a monoclonal antibody (MAb) as a probe. Nematodes, uninfected or infected with P. penetrans, were harvested at 12, 16, 24, and 38 days after inoculation (DAI) and then examined to determine the developmental stage of the bacterium. Vegetative growth of P. penetrans was observed only in infected nematodes harvested at 12 and 16 DAI, whereas cells at different stages of sporulation and mature endospores were observed at 24 and 38 DAI. ELISA and immunoblot analysis revealed that the adhesin-associated epitope was first detected at 24 DAI, and increased in the later stages of sporogenesis. These results indicate that the synthesis of adhesin-related proteins occurred at a certain developmental stage relative to the sporulation process, and was associated with endospore maturation. Immunofluorescence microscopy indicated that the distribution of the epitope is nearly uniform on the periphery of each spore, as defined by parasporal fibers. Immunocytochemistry at the ultrastructural level indicated a distribution of the epitope over the parasporal fibers. The epitope also was detected over other structures such as sporangium and exosporium during the sporogenesis process, but it was not observed over the cortex, inner-spore coat, outer-spore coat, or protoplasm. The appearance of the adhesin epitope first at stage III of sporogenesis and its presence on the parasporal fibers are consistent with an adhesin-related role in the attachment of the mature endospore to the cuticle of the nematode host.

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

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    M. Angeles López-Matas

    2013-01-01

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

  18. In Silico Perspectives on the Prediction of the PLP's Epitopes involved in Multiple Sclerosis.

    Science.gov (United States)

    Zamanzadeh, Zahra; Ataei, Mitra; Nabavi, Seyed Massood; Ahangari, Ghasem; Sadeghi, Mehdi; Sanati, Mohammad Hosein

    2017-03-01

    Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). The main cause of the MS is yet to be revealed, but the most probable theory is based on the molecular mimicry that concludes some infections in the activation of T cells against brain auto-antigens that initiate the disease cascade. The Purpose of this research is the prediction of the auto-antigen potency of the myelin proteolipid protein (PLP) in multiple sclerosis. As there wasn't any tertiary structure of PLP available in the Protein Data Bank (PDB) and in order to characterize the structural properties of the protein, we modeled this protein using prediction servers. Meta prediction method, as a new perspective in silico , was performed to fi nd PLPs epitopes. For this purpose, several T cell epitope prediction web servers were used to predict PLPs epitopes against Human Leukocyte Antigens (HLA). The overlap regions, as were predicted by most web servers were selected as immunogenic epitopes and were subjected to the BLASTP against microorganisms. Three common regions, AA 58-74 , AA 161-177 , and AA 238-254 were detected as immunodominant regions through meta-prediction. Investigating peptides with more than 50% similarity to that of candidate epitope AA 58-74 in bacteria showed a similar peptide in bacteria (mainly consistent with that of clostridium and mycobacterium) and spike protein of Alphacoronavirus 1, Canine coronavirus, and Feline coronavirus. These results suggest that cross reaction of the immune system to PLP may have originated from a bacteria or viral infection, and therefore molecular mimicry might have an important role in the progression of MS. Through reliable and accurate prediction of the consensus epitopes, it is not necessary to synthesize all PLP fragments and examine their immunogenicity experimentally ( in vitro ). In this study, the best encephalitogenic antigens were predicted based on bioinformatics tools that may provide reliable

  19. Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

    Science.gov (United States)

    Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

    2013-01-01

    The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

  20. The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.

    Directory of Open Access Journals (Sweden)

    Magdalini Polymenidou

    Full Text Available PrP(Sc, a misfolded and aggregated form of the cellular prion protein PrP(C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP(C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP(C and PrP(Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP(C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP(C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP(Sc. Other antibodies immunoprecipitate PrP(C, but not PrP(Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP(C and PrP(Sc. Amino-proximal antibodies were found to react with repetitive PrP(C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

  1. Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

    Science.gov (United States)

    Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

    2015-07-01

    Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments. © 2015 John Wiley & Sons Ltd.

  2. Allergen and Epitope Targets of Mouse-Specific T Cell Responses in Allergy and Asthma

    Directory of Open Access Journals (Sweden)

    Véronique Schulten

    2018-02-01

    Full Text Available Mouse allergy has become increasingly common, mainly affecting laboratory workers and inner-city households. To date, only one major allergen, namely Mus m 1, has been described. We sought to identify T cell targets in mouse allergic patients. PBMC from allergic donors were expanded with either murine urine or epithelial extract and subsequently screened for cytokine production (IL-5 and IFNγ in response to overlapping peptides spanning the entire Mus m 1 sequence, peptides from various Mus m 1 isoforms [major urinary proteins (MUPs], peptides from mouse orthologs of known allergens from other mammalian species and peptides from proteins identified by immunoproteomic analysis of IgE/IgG immunoblots of mouse urine and epithelial extracts. This approach let to the identification of 106 non-redundant T cell epitopes derived from 35 antigens. Three major T cell-activating regions were defined in Mus m 1 alone. Moreover, our data show that immunodominant epitopes were largely shared between Mus m 1 and other MUPs even from different species, suggesting that sequence conservation in different allergens is a determinant for immunodominance. We further identified several novel mouse T cell antigens based on their homology to known mammalian allergens. Analysis of cohort-specific T cell responses revealed that rhinitis and asthmatic patients recognized different epitope repertoires. Epitopes defined herein can be formulated into an epitope “megapool” used to diagnose mouse allergy and study mouse-specific T cell responses directly ex vivo. This analysis of T cell epitopes provides a good basis for future studies to increase our understanding of the immunopathology associated with MO-allergy and asthma.

  3. Positive-unlabeled learning for the prediction of conformational B-cell epitopes

    Science.gov (United States)

    2015-01-01

    Background The incomplete ground truth of training data of B-cell epitopes is a demanding issue in computational epitope prediction. The challenge is that only a small fraction of the surface residues of an antigen are confirmed as antigenic residues (positive training data); the remaining residues are unlabeled. As some of these uncertain residues can possibly be grouped to form novel but currently unknown epitopes, it is misguided to unanimously classify all the unlabeled residues as negative training data following the traditional supervised learning scheme. Results We propose a positive-unlabeled learning algorithm to address this problem. The key idea is to distinguish between epitope-likely residues and reliable negative residues in unlabeled data. The method has two steps: (1) identify reliable negative residues using a weighted SVM with a high recall; and (2) construct a classification model on the positive residues and the reliable negative residues. Complex-based 10-fold cross-validation was conducted to show that this method outperforms those commonly used predictors DiscoTope 2.0, ElliPro and SEPPA 2.0 in every aspect. We conducted four case studies, in which the approach was tested on antigens of West Nile virus, dihydrofolate reductase, beta-lactamase, and two Ebola antigens whose epitopes are currently unknown. All the results were assessed on a newly-established data set of antigen structures not bound by antibodies, instead of on antibody-bound antigen structures. These bound structures may contain unfair binding information such as bound-state B-factors and protrusion index which could exaggerate the epitope prediction performance. Source codes are available on request. PMID:26681157

  4. GPS-MBA: computational analysis of MHC class II epitopes in type 1 diabetes.

    Science.gov (United States)

    Cai, Ruikun; Liu, Zexian; Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu

    2012-01-01

    As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-A(g7) in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-A(g7) and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-A(g7) and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-A(g7) and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org.

  5. Comparative analysis of evolutionarily conserved motifs of epidermal growth factor receptor 2 (HER2) predicts novel potential therapeutic epitopes

    DEFF Research Database (Denmark)

    Deng, Xiaohong; Zheng, Xuxu; Yang, Huanming

    2014-01-01

    druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our...

  6. Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide

    NARCIS (Netherlands)

    Blanco, Esther; Guerra, Beatriz; Torre, de la Beatriz; Defaus, Sira; Dekker, A.; Andreu, D.; Sobrino, Francisco

    2016-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have reported (Cubillos et al., 2008) that a synthetic dendrimeric peptide consisting of four copies of a B-cell epitope [VP1(136–154)] linked through thioether bonds to a T-cell epitope [3A(21–35)

  7. [Chemical modification of allergen leading to changes in its epitopic activity].

    Science.gov (United States)

    Babakhin, A A; Gushchin, I S; Andreev, S M; Petrukhina, A I; Viler, A V; Stokinger, B; Nolte, G; Dubuske, L M; Khaitov, R M; Petrpv, R V

    1999-01-01

    Modification of a model allergen ovalbumin (OA) with succinylation led to a decrease of its allergenicity measured by passive cutaneous anaphylaxis reaction, RAST inhibition assay and basophil histamine release. Modified OA stimulated OA-specific T-cell hybrid 3DO-548 to produce IL-2 at the same level as in case of non-modified OA. Modified OA did not induce anti-OA IgE, but did induce anti-OA IgG antibodies. This approach to chemical modification of allergen-selective blockade of B-cell epitopes while not affecting T-cell epitopes suggests new opportunities in creation of safe and effective allergovaccines.

  8. Interaction of an immunodominant epitope with Ia molecules in T-cell activation

    DEFF Research Database (Denmark)

    Adorini, L; Sette, A; Buus, S

    1988-01-01

    The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis o......-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity....

  9. Bioinformatics prediction of swine MHC class I epitopes from Porcine Reproductive and Respiratory Syndrome Virus

    DEFF Research Database (Denmark)

    Welner, Simon; Nielsen, Morten; Lund, Ole

    an effective CTL response against PRRSV, we have taken a bioinformatics approach to identify common PRRSV epitopes predicted to react broadly with predominant swine MHC (SLA) alleles. First, the genomic integrity and sequencing method was examined for 334 available complete PRRSV type 2 genomes leaving 104...... by the PopCover algorithm, providing a final list of 54 epitopes prioritized according to maximum coverage of PRRSV strains and SLA alleles. This bioinformatics approach provides a rational strategy for selecting peptides for a CTL-activating vaccine with broad coverage of both virus and swine diversity...

  10. Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.

    Science.gov (United States)

    de Lalla, C; Sturniolo, T; Abbruzzese, L; Hammer, J; Sidoli, A; Sinigaglia, F; Panina-Bordignon, P

    1999-08-15

    Although atopic allergy affects Lol p5a allergen from rye grass. In vitro binding studies confirmed the promiscuous binding characteristics of these peptides. Moreover, most of the predicted ligands were novel T cell epitopes that were able to stimulate T cells from atopic patients. We generated a panel of Lol p5a-specific T cell clones, the majority of which recognized the peptides in a cross-reactive fashion. The computational prediction of DR ligands might thus allow the design of T cell epitopes with potential useful application in novel immunotherapy strategies.

  11. Hepatitis C virus epitope exposure and neutralization by antibodies is affected by time and temperature

    DEFF Research Database (Denmark)

    Sabo, Michelle C; Luca, Vincent C; Ray, Stuart C

    2012-01-01

    A recent study with flaviviruses suggested that structural dynamics of the virion impact antibody neutralization via exposure of ostensibly cryptic epitopes. To determine whether this holds true for the distantly related hepatitis C virus (HCV), whose neutralizing epitopes may be obscured...... by a glycan shield, apolipoprotein interactions, and the hypervariable region on the E2 envelope protein, we assessed how time and temperature of pre-incubation altered monoclonal antibody (MAb) neutralization of HCV. Notably, several MAbs showed increased inhibitory activity when pre-binding was performed...

  12. The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity.

    Science.gov (United States)

    Wakata, Aika; Kanemoto, Satoshi; Tang, Huamin; Kawabata, Akiko; Nishimura, Mitsuhiro; Jasirwan, Chyntia; Mahmoud, Nora Fahmy; Mori, Yasuko

    2018-03-01

    Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity. IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents

  13. MuPeXI: prediction of neo-epitopes from tumor sequencing data

    DEFF Research Database (Denmark)

    Bjerregaard, Anne-Mette; Nielsen, Morten; Hadrup, Sine Reker

    2017-01-01

    Personalization of immunotherapies such as cancer vaccines and adoptive T cell therapy depends on identification of patient-specific neo-epitopes that can be specifically targeted. MuPeXI, the mutant peptide extractor and informer, is a program to identify tumor-specific peptides and assess...... their potential to be neo-epitopes. The program input is a file with somatic mutation calls, a list of HLA types, and optionally a gene expression profile. The output is a table with all tumor-specific peptides derived from nucleotide substitutions, insertions, and deletions, along with comprehensive annotation...

  14. Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Sørensen, Rikke Bæk; Ritter, Cathrin

    2011-01-01

    . Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition......, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion......, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors....

  15. Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Sørensen, Rikke Bæk; Ritter, Cathrin

    2011-01-01

    . Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-¿ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition......, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion......, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors....

  16. A Recombinant HAV Expressing a Neutralization Epitope of HEV Induces Immune Response against HAV and HEV in Mice.

    Science.gov (United States)

    Xiang, Kui; Kusov, Yuri; Ying, Guan; Yan, Wang; Shan, Yi; Jinyuan, Wu; Na, Yin; Yan, Zhou; Hongjun, Li; Maosheng, Sun

    2017-09-15

    Hepatitis A virus (HAV) and hepatitis E virus (HEV) are causative agents of acute viral hepatitis transmitted via the fecal-oral route. Both viruses place a heavy burden on the public health and economy of developing countries. To test the possibility that HAV could be used as an expression vector for the development of a combination vaccine against hepatitis A and E infections, recombinant HAV-HEp148 was created as a vector to express an HEV neutralization epitope (HEp148) located at aa 459-606 of the HEV capsid protein. The recombinant virus expressed the HEp148 protein in a partially dimerized state in HAV-susceptible cells. Immunization with the HAV-HEp148 virus induced a strong HAV- and HEV-specific immune response in mice. Thus, the present study demonstrates a novel approach to the development of a combined hepatitis A and E vaccine.

  17. Characterization of an extracellular epitope antibody to the neuronal K-Cl cotransporter, KCC2.

    Science.gov (United States)

    Gagnon, Kenneth Be; Fyffe, Robert Ew; Adragna, Norma C; Lauf, Peter K

    2007-07-01

    1. Ion gradients across the cell membrane are important for proper cellular communication and homeostasis. With the exception of erythrocytes, chloride (Cl), one of the most important free anions in animal cells, is not distributed at thermodynamic equilibrium across the plasma membrane. The K-Cl cotransporter (COT), consisting of at least four isoforms, utilizes the larger outwardly directed chemical driving force of K to expel Cl from the cell against its inwardly directed chemical gradient and has been implicated recently as one of the main Cl extruders in developing neurons. 2. Previous in situ hybridization studies have indicated widespread mRNA distribution of the neuronal-specific K-Cl COT isoform (KCC2) throughout the rat central nervous system (CNS). However, immunohistochemical studies have been limited owing to the availability of a more selective antibody to KCC2. The goal of the present study was to develop a new molecular tool for the immunohistochemical identification and neuronal distribution of KCC2. 3. Herein, we present evidence of immunohistochemical corroboration of the widespread KCC2 mRNA expression using a novel extracellular anti-peptide antibody directed against the second extracellular loop (ECL2) of KCC2. Immunoperoxidase and immunofluorescent labelling revealed widespread post-synaptic somatic and dendritic localization of KCC2 in multiple neuronal populations in the cerebral cortex, hippocampus, brainstem, lumbar spinal cord and cerebellum. We also demonstrate that binding of the antibody to an extracellular epitope within ECL2 does not alter cotransporter function. In essence, the present study reports on a new molecular tool for structural and functional studies of KCC2.

  18. Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.

    Directory of Open Access Journals (Sweden)

    Paul Thiamjoo Tan

    2010-01-01

    Full Text Available 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.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-gamma 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.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.

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

    Science.gov (United States)

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

    2018-04-26

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

  20. Delivery presentations

    Science.gov (United States)

    Pregnancy - delivery presentation; Labor - delivery presentation; Occiput posterior; Occiput anterior; Brow presentation ... The mother can walk, rock, and try different delivery positions during labor to help encourage the baby ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Pierce, Brian G.; Boucher, Elisabeth N.; Piepenbrink, Kurt H.; Ejemel, Monir; Rapp, Chelsea A.; Thomas, William D.; Sundberg, Eric J.; Weng, Zhiping; Wang, Yang; Diamond, Michael S.

    2017-08-09

    Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines.

    IMPORTANCEHepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that

  2. Identification of novel HLA-A(*)0201-restricted CTL epitopes from Pokemon.

    Science.gov (United States)

    Yuan, Bangqing; Zhao, Lin; Xian, Ronghua; Zhao, Gang

    2012-01-01

    Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. A dominant EV71-specific CD4+ T cell epitope is highly conserved among human enteroviruses.

    Directory of Open Access Journals (Sweden)

    Ruicheng Wei

    Full Text Available CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD, has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.

  4. Factor H and Properdin Recognize Different Epitopes on Renal Tubular Epithelial Heparan Sulfate

    NARCIS (Netherlands)

    Zaferani, Azadeh; Vives, Romain R.; van der Pol, Pieter; Navis, Gerjan J.; Daha, Mohamed R.; van Kooten, Cees; Lortat-Jacob, Hugues; Seelen, Marc A.; van den Born, Jacob

    2012-01-01

    During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope

  5. Characterization of binding specificities of bovine leucocyte class I molecules: impacts for rational epitope discovery

    DEFF Research Database (Denmark)

    Hansen, Andreas M.; Rasmussen, Michael; Svitek, Nicholas

    2014-01-01

    confirmed experimentally. This study demonstrates how biochemical high-throughput assays combined with immunoinformatics can be used to characterize the peptide-binding motifs of BoLA-I molecules, boosting performance of MHC peptide-binding prediction methods, and empowering rational epitope discovery...

  6. Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope

    DEFF Research Database (Denmark)

    Iversen, Astrid K N; Stewart-Jones, Guillaume; Learn, Gerald H

    2006-01-01

    two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which...

  7. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  8. Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools

    DEFF Research Database (Denmark)

    Greenbaum, Jason A.; Andersen, Pernille; Blythe, Martin

    2007-01-01

    and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington...

  9. Label free targeted detection and quantification of celiac disease immunogenic epitopes by mass spectrometry

    NARCIS (Netherlands)

    Broeck, van den H.C.; Cordewener, J.H.G.; Nessen, M.A.; America, A.H.P.; Meer, van der I.M.

    2015-01-01

    Celiac disease (CD) is a food-related disease caused by certain gluten peptides containing T-cell stimulating epitopes from wheat, rye, and barley. CD-patients have to maintain a gluten-free diet and are therefore dependent on reliable testing and labeling of gluten-free products. So far, the

  10. DPD epitope-specific glutamic acid decarboxylase GAD)65 autoantibodies in children with Type 1 diabetes

    Science.gov (United States)

    To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical dat...

  11. Characterisation of a protective linear B cell epitope against feline parvoviruses

    NARCIS (Netherlands)

    Langeveld, J.P.; Martinez Torrecuadrada, J.; Boshuizen, R.S.; Meloen, R.H.; Ignacio Casal, J.

    2001-01-01

    Monoclonal antibody 3C9 was the starting material in the definition of the epitope that led to the synthesis of the first efficient peptide vaccine against a viral disease (canine parvovirus) in the natural host (dog). In this report, we have analysed the specificity of the antibody at the single

  12. Conservation of HIV-1 T cell epitopes across time and clades

    DEFF Research Database (Denmark)

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou

    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 immunoinfor...

  13. State of the art and challenges in sequence based T-cell epitope prediction

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Hoof, Ilka; Lund, Ole

    2010-01-01

    Sequence based T-cell epitope predictions have improved immensely in the last decade. From predictions of peptide binding to major histocompatibility complex molecules with moderate accuracy, limited allele coverage, and no good estimates of the other events in the antigen-processing pathway, the...

  14. The design and implementation of the immune epitope database and analysis resource

    DEFF Research Database (Denmark)

    Peters, B.; Sidney, J.; Bourne, P.

    2005-01-01

    Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is r...

  15. Thyroid Autoantibodies Display both “Original Antigenic Sin” and Epitope Spreading

    OpenAIRE

    McLachlan, Sandra M.; Rapoport, Basil

    2017-01-01

    Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyrotropin receptor (TSHR) A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of...

  16. Key residues of a major cytochrome P4502D6 epitope are located on the surface of the molecule.

    Science.gov (United States)

    Ma, Yun; Thomas, Mark G; Okamoto, Manabu; Bogdanos, Dimitrios P; Nagl, Sylvia; Kerkar, Nanda; Lopes, Agnel R; Muratori, Luigi; Lenzi, Marco; Bianchi, Francesco B; Mieli-Vergani, Giorgina; Vergani, Diego

    2002-07-01

    Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1-193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267-337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6(316-327) is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.

  17. IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Nielsen, H.; Eiwegger, T.

    2014-01-01

    epitopes. Objective: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. Methods: Polyclonal sera from three individual patients......, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library...

  18. Tat protein vaccination of cynomolgus macaques influences SHIV-89.6P cy243 epitope variability.

    Science.gov (United States)

    Ridolfi, Barbara; Genovese, Domenico; Argentini, Claudio; Maggiorella, Maria Teresa; Sernicola, Leonardo; Buttò, Stefano; Titti, Fausto; Borsetti, Alessandra; Ensoli, Barbara

    2008-02-01

    In a previous study we showed that vaccination with the native Tat protein controlled virus replication in five out of seven monkeys against challenge with the simian human immunodeficiency virus (SHIV)-89.6P cy243 and that this protection correlated with T helper (Th)-1 response and cytotoxic T lymphocyte (CTL) activity. To address the evolution of the SHIV-89.6P cy243 both in control and vaccinated infected monkeys, the sequence of the human immunodeficiency virus (HIV)-1 Tat protein and the C2-V3 Env region of the proviral-DNA-derived clones were analyzed in both control and vaccinated but unprotected animals. We also performed analysis of the T cell epitope using a predictive epitope model taking into consideration the phylogeny of the variants. Our results suggest that even though the viral evolution observed in both groups of monkeys was directed toward variations in the major histocompatibility complex (MHC)-I epitopes, in the control animals it was associated with mutational escape of such epitopes. On the contrary, it is possible that viral evolution in the vaccinated monkeys was linked to mutations that arose to keep high the viral fitness. In the vaccinated animals the reduction of epitope variability, obtained prompting the immune system by vaccination and inducing a specific immunological response against virus, was able to reduce the emergence of escape mutants. Thus the intervention of host's selective forces in driving CTL escape mutants and in modulating viral fitness appeared to be different in the two groups of monkeys. We concluded that in the vaccinated unprotected animals, vaccination with the Tat protein induced a broad antiviral response, as demonstrated by the reduced ability to develop escape mutants, which is known to help in the control of viral replication.

  19. Mapping of Minimal Motifs of B-Cell Epitopes on Human Zona Pellucida Glycoprotein-3

    Directory of Open Access Journals (Sweden)

    Wan-Xiang Xu

    2012-01-01

    Full Text Available The human zona pellucida glycoprotein-3 (hZP3 by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3, two predictable epitopes23–30  and  301–320 on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a22–176 or hZP3b177–348 as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL23–28 for hZP323–30, MQVTDD103–108 for hZP393–110, EENW178–181 for hZP3172–190, as well as SNSWF306–310 and EGP313–315 for hZP3301–320, respectively. Furthermore, the antigenicity of two peptides for hZP3172–187 and hZP3301–315 and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b177–348 protein, as well as r-hZP3172–190, r-hZP3303–310, and r-hZP3313–320 epitope peptides fused with truncated GST188 protein.

  20. Epitope mapping of monoclonal antibodies directed to aminopeptidase A and their relevance for albuminuria in mice.

    Science.gov (United States)

    Gerlofs-Nijland, Miriam E; Assmann, Karel J M; van Son, Jacco P H F; Dijkman, Henry B P M; te Loeke, Nathalie A J M; van der Zee, Ruurd; Wetzels, Jack F M; Groenen, Patricia J T A

    2003-01-01

    We have shown previously that injection of specific combinations of anti-aminopeptidase A monoclonal antibodies induces an acute massive albuminuria in mice. This albuminuria is neither dependent on systemic mediators of inflammation nor angiotensin II. In this study, we examined the contribution of two individual antibodies, the enzyme-inhibiting antibody ASD-37 and the non-enzyme-inhibiting antibody ASD-41, in the induction of albuminuria as well as the interactions between these two monoclonals. In addition, we have mapped the epitopes of both antibodies using in vitro coupled transcription/translation of specifically designed cDNA fragments followed by immunoprecipitation, and using peptide enzyme-linked immunosorbent assay in case of a continuous epitope. A single intravenous injection of 4 mg of either ASD-37 or ASD-41 did not induce albuminuria. This dose of ASD-37 did not completely inhibit enzyme activity. The combination of 4 mg ASD-37/41 (1:1 weight ratio) induced albuminuria and almost completely inhibited enzyme activity. Similar results were obtained with a combination of ASD-37/41 in a 1:39 or 39:1 weight ratio. Administration of 2 mg ASD-41 24 h before injection of 2 mg ASD-37 significantly enhanced albuminuria. The epitope of ASD-37 is located at the C-terminal end of aminopeptidase A, whereas the ASD-41 epitope is mapped near the enzyme active site. Our data suggest that ASD-41 modulates the binding of ASD-37 to its epitope and/or vice versa. As a consequence, ASD-37 and ASD-41 act synergistically, not only in inhibiting enzyme activity but also in inducing albuminuria. Copyright 2003 S. Karger AG, Basel

  1. Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

    Science.gov (United States)

    Abana, Chike O; Pilkinton, Mark A; Gaudieri, Silvana; Chopra, Abha; McDonnell, Wyatt J; Wanjalla, Celestine; Barnett, Louise; Gangula, Rama; Hager, Cindy; Jung, Dae K; Engelhardt, Brian G; Jagasia, Madan H; Klenerman, Paul; Phillips, Elizabeth J; Koelle, David M; Kalams, Spyros A; Mallal, Simon A

    2017-11-01

    Select CMV epitopes drive life-long CD8 + T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4 + T cells specific for human CMV (HCMV) are elevated in HIV + HCMV + subjects. To determine whether HCMV epitope-specific CD4 + T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4 + T cells in coinfected HLA-DR7 + long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4 + T cells were inflated among these HIV + subjects compared with those from an HIV - HCMV + HLA-DR7 + cohort or with HLA-DR7-restricted CD4 + T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4 + T cells consisted of effector memory or effector memory-RA + subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX 3 CR1, CD38, or HLA-DR but less often coexpressed CD38 + and HLA-DR + The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4 + T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease. Copyright © 2017 by The American Association of Immunologists, Inc.

  2. Identification of two novel rabbit hemorrhagic disease virus (RHDV) B cell epitopes and evaluation of its immunoprotection against RHDV.

    Science.gov (United States)

    DeSheng, Kong; HuaiRan, Liu; JiaSen, Liu; Zuo, Yu; Qian, Jiang; DongChun, Guo; XiaoLiang, Hu; FengJie, Wang; QianQian, Huang; LianDong, Qu

    2015-07-01

    The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.

  3. Glutamic acid decarboxylase-derived epitopes with specific domains expand CD4(+CD25(+ regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Guojiang Chen

    Full Text Available BACKGROUND: CD4(+CD25(+ regulatory T cell (Treg-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. METHODOLOGY/PRINCIPAL FINDINGS: Splenic lymphocytes from nonobese diabetic (NOD mice were stimulated with different glutamic acid decarboxylase (GAD-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538-expanded CD4(+CD25(+ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528- or p530 (GAD530-543-expanded CD4(+CD25(+ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes between the above-mentioned epitopes and MHC class II I-A(g7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7. Furthermore, p524 bound to I-A(g7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570, which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+CD25(+ T cells suppressed the onset of diabetes in NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of

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

    DEFF Research Database (Denmark)

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

    2017-01-01

    and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation....... The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots...

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

    Science.gov (United States)

    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

  6. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus

    2011-01-01

    , the functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  7. GAKG-RGEKG an Epitope That Provokes Immune Cross-Reactivity between Prevotella sp. and Human Collagen: Evidence of Molecular Mimicry in Chronic Periodontitis

    Directory of Open Access Journals (Sweden)

    Gustavo Alberto Obando-Pereda

    2016-01-01

    Full Text Available Periodontal disease afflicts 20% of world population. This process usually occurs in the form of being lethargic and chronic, and consequently this disease is known as chronic process. All chronic diseases constantly cause activation of the immune system, and therefore the presentation of microbial peptides which are presented to lymphocytes by professional antigen presenting cells can present microbial peptides very similar to important structures of human economy causing autoimmune diseases, process known as molecular mimicry. Thus, the aim of this study was to verify the presence of molecular mimicry phenomenon between periodontopathogens and human proteins. Blasting microbes of Socransky periodontal complexes against human collagen were performed and then the proteins with similarities were modelled and were screened in the MHI binding virtual methods. The epitopes selected were produced and plasma of chronic periodontal volunteers was obtained and a dot immunobinding assay was performed. Hypothetical protein of Prevotella sp. and human collagen epitopes with high similarities were positive for dot immunobinding assay. With this result it can be suggested that the mimicry phenomena can occur on periodontal disease.

  8. Lecture Presentations

    International Nuclear Information System (INIS)

    2007-01-01

    The Heavy-Ion Collisions in the LHC workshop held in Cracow from 18 to 18 May 2007. The main subject of the workshop was to present the newest results of research provided at CERN LHC collider. Additionally some theoretical models and methods used for presented data analysis were discussed

  9. CATCHY PRESENTATIONS

    DEFF Research Database (Denmark)

    Eriksen, Kaare; Tollestrup, Christian; Ovesen, Nis

    2011-01-01

    An important competence for designers is the ability to communicate and present ideas and proposals for customers, partners, investors and colleagues. The Pecha Kucha principle, developed by Astrid Klein and Mark Dytham, has become a widely used and easy format for the presentation of new concepts...

  10. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    Science.gov (United States)

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Workshop presentations

    International Nuclear Information System (INIS)

    Sanden, Per-Olof; Edland, Anne; Reiersen, Craig; Mullins, Peter; Ingemarsson, Karl-Fredrik; Bouchard, Andre; Watts, Germaine; Johnstone, John; Hollnagel, Erik; Ramberg, Patric; Reiman, Teemu

    2009-01-01

    An important part of the workshop was a series of invited presentations. The presentations were intended to both provide the participants with an understanding of various organisational approaches and activities as well as to stimulate the exchange of ideas during the small group discussion sessions. The presentation subjects ranged from current organisational regulations and licensee activities to new organisational research and the benefits of viewing organisations from a different perspective. There were more than a dozen invited presentations. The initial set of presentations gave the participants an overview of the background, structure, and aims of the workshop. This included a short presentation on the results from the regulatory responses to the pre-workshop survey. Representatives from four countries (Sweden, Canada, Finland, and the United Kingdom) expanded upon their survey responses with detailed presentations on both regulatory and licensee safety-related organisational activities in their countries. There were also presentations on new research concerning how to evaluate safety critical organisations and on a resilience engineering perspective to safety critical organisations. Below is the list of the presentations, the slides of which being available in Appendix 2: 1 - Workshop Welcome (Per-Olof Sanden); 2 - CSNI Working Group on Human and Organisational Factors (Craig Reiersen); 3 - Regulatory expectations on justification of suitability of licensee organisational structures, resources and competencies (Anne Edland); 4 - Justifying the suitability of licensee organisational structures, resources and competencies (Karl-Fredrik Ingemarsson); 5 - Nuclear Organisational Suitability in Canada (Andre Bouchard); 6 - Designing and Resourcing for Safety and Effectiveness (Germaine Watts); 7 - Organisational Suitability - What do you need and how do you know that you've got it? (Craig Reiersen); 8 - Suitability of Organisations - UK Regulator's View (Peter

  12. CERN presentations

    CERN Multimedia

    CERN. Geneva

    2011-01-01

    Presentation by CERN (10 minutes each) Rolf Landua - Education and Outreach Salvatore Mele - Open Access Jean-Yves Le Meur - Digital Library in Africa Francois Fluckiger - Open Source/Standards (tbc) Tim Smith - Open Data for Science Tullio Basiglia - tbc

  13. Delivery presentations

    Science.gov (United States)

    ... has entered the pelvis. If the presenting part lies above the ischial spines, the station is reported ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  14. Information Presentation

    Science.gov (United States)

    Holden, K.L.; Boyer, J.L.; Sandor, A.; Thompson, S.G.; McCann, R.S.; Begault, D.R.; Adelstein, B.D.; Beutter, B.R.; Stone, L.S.

    2009-01-01

    The goal of the Information Presentation Directed Research Project (DRP) is to address design questions related to the presentation of information to the crew. The major areas of work, or subtasks, within this DRP are: 1) Displays, 2) Controls, 3) Electronic Procedures and Fault Management, and 4) Human Performance Modeling. This DRP is a collaborative effort between researchers at Johnson Space Center and Ames Research Center.

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

    Directory of Open Access Journals (Sweden)

    Christina Gerstner

    2016-11-01

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

  16. Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.

    Science.gov (United States)

    Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J

    2011-03-01

    Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.

  17. Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

    Science.gov (United States)

    Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

    1998-06-01

    We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

  18. Identifikasi epitop dari Streptococcus mutans terhadap sekretori Imunoglobulin A saliva (The identification of Streptococcus mutans epitopes to secretory Immunoglobulin A saliva

    Directory of Open Access Journals (Sweden)

    Anita Yuliati

    2005-09-01

    Full Text Available S. mutans is one of the etiology agent of dental caries, these bacteria have a surface protein of about 185 kDa named Ag I/II. The secretory of sIgA saliva to Ag I/II of S.mutans has shown to be able to prevent colonization in human oral cavity. Peptides derived from the 824 to 853 residues of the P region of antigen I/II S. mutans related to the pathogenesis of dental caries. The aim of this study was to identify the overlapping sequence of amino acids (epitope derived from the 624 to 853 residues of P of antigen I/II S. mutans to sIgA saliva on caries and caries-free subject in a observational cross sectional study. The P region of antigen I/II S.mutans was cut into 22 peptides of 9 mer sequences with an overlapping of 8 mer and an offset of 1 mer, synthesized on polyethylene pins and tested for the reactivity with an ELISA indirect method to sIgA saliva on caries and caries-free subject. The results of this study showed that amino acid sequences with TPPVKP (832–837 and TAPTKPTY (838–845 were reactive to sIgA saliva on caries and caries-free subject. The conclusion of this study was that the overlapping common sequence of amino acid (epitopes corresponding to TPPVKP (832–837 and TAPTKPTY (838–845 was identified as caries marker epitopes in human.

  19. Molecular mass distribution and epitopes of the beta lactoglobulin submitted to hydrolysis pre-transglutaminase treatment

    International Nuclear Information System (INIS)

    Villas-Boas, M.B.; Zollner, R.L.; Netto, F.M.; Paes Leme, A.F.; Benede, S.; Molina, E.

    2012-01-01

    Full text: The β-Lactoglobulin (β-Lg) is a whey protein with important nutritional proper ties but very resistant to pepsin digestion and consequently highly antigenic. This protein can be modified by transglutaminase (TG) although it is required a pretreatment to increase their susceptibility to the TG action. In the present study the hydrolysis pre-TG treatment was used to improve the TG accessibility on β-Lg and the MM distribution and antigenic fragments were evaluated. For pre-TG treatment, the β-Lg (Davisco Inc.) was hydrolyzed with bromelain (3% of β-Lg w/w in distilled water; 25 U enzyme g 1 of substrate, pH 7.5, 240 min) and then polymerized by TG (7% hydrolysate, 10U TG g 1 protein, 50 C/180 min). The samples were evaluated by SDS-PAGE/tricine and by RP-nanoUPLC (nanoAcquity UPLC, Waters) coupled with nano-electrospray tandem mass spectrometry on a Q-Tof Ultima API mass spectrometer (MicroMass/Waters) at LNBio. The products were also submitted to pepsin digestion and the peptide identification was performed by RP-HPLC-tandem mass spectrometry (RP-HPLC-MS/MS, Brucker) with support from CIAL. The β-Lg hydrolysed by bromelain and polymerized by TG had a broad MM distribution. The intact mass analysis indicated that the non modified βLg -A showed 18.362 Da and the non modified βLg -B 18.274 Da, which is in agreement with the theoretical corresponding masses. The use of bromelain pre-TG treatment resulted in polymers with MM from 61.052 to 67.654 Da, although some non modified protein was still present. In addition, the non modified β-Lg showed fragments that present high antigenicity (such as Leu 95 - Leu 104 , Asp 95 - Phe 105 , Tyr 42 - Leu 54 , lle 29 - Val 41 ), previously identified as IgE-binding epitopes. After hydrolysis following by TG treatment the fragment Tyr 42 - Leu 54 was still present, however the other fragments that were observed in the non modified β-Lg were not detected by LC-MS/MS, suggesting that structural change occurred in

  20. Molecular mass distribution and epitopes of the beta lactoglobulin submitted to hydrolysis pre-transglutaminase treatment

    Energy Technology Data Exchange (ETDEWEB)

    Villas-Boas, M.B.; Zollner, R.L.; Netto, F.M. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil); Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Benede, S.; Molina, E. [Universidad Autonoma de Madrid (Spain)

    2012-07-01

    Full text: The {beta}-Lactoglobulin ({beta}-Lg) is a whey protein with important nutritional proper ties but very resistant to pepsin digestion and consequently highly antigenic. This protein can be modified by transglutaminase (TG) although it is required a pretreatment to increase their susceptibility to the TG action. In the present study the hydrolysis pre-TG treatment was used to improve the TG accessibility on {beta}-Lg and the MM distribution and antigenic fragments were evaluated. For pre-TG treatment, the {beta}-Lg (Davisco Inc.) was hydrolyzed with bromelain (3% of {beta}-Lg w/w in distilled water; 25 U enzyme g{sup 1} of substrate, pH 7.5, 240 min) and then polymerized by TG (7% hydrolysate, 10U TG g{sup 1} protein, 50 C/180 min). The samples were evaluated by SDS-PAGE/tricine and by RP-nanoUPLC (nanoAcquity UPLC, Waters) coupled with nano-electrospray tandem mass spectrometry on a Q-Tof Ultima API mass spectrometer (MicroMass/Waters) at LNBio. The products were also submitted to pepsin digestion and the peptide identification was performed by RP-HPLC-tandem mass spectrometry (RP-HPLC-MS/MS, Brucker) with support from CIAL. The {beta}-Lg hydrolysed by bromelain and polymerized by TG had a broad MM distribution. The intact mass analysis indicated that the non modified {beta}Lg -A showed 18.362 Da and the non modified {beta}Lg -B 18.274 Da, which is in agreement with the theoretical corresponding masses. The use of bromelain pre-TG treatment resulted in polymers with MM from 61.052 to 67.654 Da, although some non modified protein was still present. In addition, the non modified {beta}-Lg showed fragments that present high antigenicity (such as Leu{sub 95} - Leu{sub 104}, Asp{sub 95} - Phe{sub 105}, Tyr{sub 42} - Leu{sub 54}, lle{sub 29} - Val{sub 41}), previously identified as IgE-binding epitopes. After hydrolysis following by TG treatment the fragment Tyr{sub 42} - Leu{sub 54} was still present, however the other fragments that were observed in the non

  1. Conserved B-cell epitopes among human bocavirus species indicate potential diagnostic targets.

    Directory of Open Access Journals (Sweden)

    Zhuo Zhou

    Full Text Available 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.

  2. Voting Present

    Directory of Open Access Journals (Sweden)

    James Lo

    2013-12-01

    Full Text Available During his time as a state senator in Illinois, Barack Obama voted “Present” 129 times, a deliberate act of nonvoting that subsequently became an important campaign issue during the 2008 presidential elections. In this article, I examine the use of Present votes in the Illinois state senate. I find evidence that Present votes can largely be characterized as protest votes used as a legislative tool by the minority party. Incorporating information from Present votes into a Bayesian polytomous item-response model, I find that this information increases the efficiency of ideal point estimates by approximately 35%. There is little evidence of significant moderation by Obama when Present votes are accounted for, though my results suggest that Obama’s voting record may have moderated significantly before his subsequent election to the U.S. Senate. My results also suggest that because legislative nonvoting may occur for a variety of reasons, naive inclusion of nonvoting behavior into vote choice models may lead to biased results.

  3. Lecture Presentations

    International Nuclear Information System (INIS)

    2008-01-01

    The Symposium on Physics of Elementary Interactions in the LHC Era held in Warsaw from 21 to 22 April 2008. The main subject of the workshop was to present the progress in CERN LHC collider project. Additionally some satellite activities in field of education, knowledge and technology transfer in the frame of CERN - Poland cooperation were shown

  4. Presentation Technique

    International Nuclear Information System (INIS)

    Froejmark, M.

    1992-10-01

    The report presents a wide, easily understandable description of presentation technique and man-machine communication. General fundamentals for the man-machine interface are illustrated, and the factors that affect the interface are described. A model is presented for describing the operators work situation, based on three different levels in the operators behaviour. The operator reacts routinely in the face of simple, known problems, and reacts in accordance with predetermined plans in the face of more complex, recognizable problems. Deep fundamental knowledge is necessary for truly complex questions. Today's technical status and future development have been studied. In the future, the operator interface will be based on standard software. Functions such as zooming, integration of video pictures, and sound reproduction will become common. Video walls may be expected to come into use in situations in which several persons simultaneously need access to the same information. A summary of the fundamental rules for the design of good picture ergonomics and design requirements for control rooms are included in the report. In conclusion, the report describes a presentation technique within the Distribution Automation and Demand Side Management area and analyses the know-how requirements within Vattenfall. If different systems are integrated, such as geographical information systems and operation monitoring systems, strict demands are made on the expertise of the users for achieving a user-friendly technique which is matched to the needs of the human being. (3 figs.)

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

    Science.gov (United States)

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

    2010-06-01

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

  6. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

    Directory of Open Access Journals (Sweden)

    Rasool Suhail

    2007-09-01

    II diabetes, demonstrating its generic specificity for amyloid fibrils. Conclusion Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

  7. Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV.

    Directory of Open Access Journals (Sweden)

    Christopher Chiu

    2014-03-01

    Full Text Available Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV, we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1 along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.

  8. Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes

    OpenAIRE

    Mazor, Ronit; Eberle, Jaime A.; Hu, Xiaobo; Vassall, Aaron N.; Onda, Masanori; Beers, Richard; Lee, Elizabeth C.; Kreitman, Robert J.; Lee, Byungkook; Baker, David; King, Chris; Hassan, Raffit; Benhar, Itai; Pastan, Ira

    2014-01-01

    Recombinant immunotoxins have produced complete remissions in leukemia patients where many doses can be given but are less active in patients with solid tumors because their immune system makes antidrug antibodies, which inactivate the immunotoxin. To suppress the immune response, we have identified and largely silenced the T-cell epitopes responsible for the immune response. A redesigned immunotoxin with T-cell epitope mutations is highly cytotoxic to cell lines and to cells isolated from ca...

  9. Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

    Czech Academy of Sciences Publication Activity Database

    Vaculík, Petr; Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Čeřovská, Noemi; Šmahel, M.

    2015-01-01

    Roč. 120, č. 2 (2015), s. 671-680 ISSN 0167-6857 R&D Projects: GA ČR(CZ) GAP501/12/1761 Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Potato virus X * Human papillomavirus * Nicotiana benthamiana Subject RIV: EE - Microbiology, Virology Impact factor: 2.390, year: 2015

  10. Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes.

    Science.gov (United States)

    Setz, Christian; Friedrich, Melanie; Rauch, Pia; Fraedrich, Kirsten; Matthaei, Alina; Traxdorf, Maximilian; Schubert, Ulrich

    2017-08-12

    In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)-the proteasome holoenzymes and a number of ubiquitin ligases-play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1 NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells.

  11. Technical presentation

    CERN Document Server

    FI Department

    2008-01-01

    RADIOSPARES, the leading catalogue distributor of components (electronic, electrical, automation, etc.) and industrial supplies will be at CERN on Friday 3 October 2008 (Main Building, Room B, from 9.00 a.m. to 3.00 p.m.) to introduce its new 2008/2009 catalogue. This will be the opportunity for us to present our complete range of products in more detail: 400 000 part numbers available on our web site (Radiospares France, RS International, extended range of components from other manufacturers); our new services: quotations, search for products not included in the catalogue, SBP products (Small Batch Production: packaging in quantities adapted to customers’ requirements); partnership with our focus manufacturers; demonstration of the on-line purchasing tool implemented on our web site in conjunction with CERN. RADIOSPARES will be accompanied by representatives of FLUKE and TYCO ELECTRONICS, who will make presentations, demonstrate materials and answer any technical questio...

  12. Evaluating the Immunogenicity of Protein Drugs by Applying In Vitro MHC Binding Data and the Immune Epitope Database and Analysis Resource

    Directory of Open Access Journals (Sweden)

    Sinu Paul

    2013-01-01

    Full Text Available The immune system has evolved to become highly specialized in recognizing and responding to pathogens and foreign molecules. Specifically, the function of HLA class II is to ensure that a sufficient sample of peptides derived from foreign molecules is presented to T cells. This leads to an important concern in human drug development as the possible immunogenicity of biopharmaceuticals, especially those intended for chronic administration, can lead to reduced efficacy and an undesired safety profile for biological therapeutics. As part of this review, we will highlight the molecular basis of antigen presentation as a key step in the induction of T cell responses, emphasizing the events associated with peptide binding to polymorphic and polygenic HLA class II molecules. We will further review methodologies that predict HLA class II binding peptides and candidate epitopes. We will focus on tools provided by the Immune Epitope Database and Analysis Resource, discussing the basic features of different prediction methods, the objective evaluation of prediction quality, and general guidelines for practical use of these tools. Finally the use, advantages, and limitations of the methodology will be demonstrated in a review of two previous studies investigating the immunogenicity of erythropoietin and timothy grass pollen.

  13. Prediction of T-cell Epitopes for Therapeutic and Prophylactic Vaccines

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby

    2007-01-01

    : The bacteria Mycobacterium tuberculosis, Influenza A virus, HIV, Yellow fever virus, and West Nile virus. For each of the above-mentioned viruses, a number of predicted CTL epitopes was subsequently selected in such a way that they together constitute a broad coverage of the available viral strains. Part IV......The spread of existing infectious diseases and the emergence of new ones call for efficient methods for vaccine development. Some of the important players in conferring immunity against pathogens are the Cytotoxic T Lymphocytes (CTL), which eliminate infected cells. Due to their deleterious effects...... vaccine design as well as for diagnostic purposes and is the centre of focus of this thesis: Part I of the thesis is an introduction to the field. In part II, I describe how we generated a method, NetCTL, for predicting CTL epitopes by integrating existing methods for predicting proteasomal cleavage, TAP...

  14. Microarray Glycan Profiling Reveals Algal Fucoidan Epitopes in Diverse Marine Metazoans

    Directory of Open Access Journals (Sweden)

    Armando A. Salmeán

    2017-09-01

    Full Text Available Despite the biological importance and pharmacological potential of glycans from marine organisms, there are many unanswered questions regarding their distribution, function, and evolution. Here we describe microarray-based glycan profiling of a diverse selection of marine animals using antibodies raised against fucoidan isolated from a brown alga. We demonstrate the presence of two fucoidan epitopes in six animals belonging to three phyla including Porifera, Molusca, and Chordata. We studied the spatial distribution of these epitopes in Cliona celata (“boring sponge” and identified their restricted localization on the surface of internal chambers. Our results show the potential of high-throughput screening and probes commonly used in plant and algal cell wall biology to study the diversity and distribution of glycan structures in metazoans.

  15. Mapping of Epitopes Occurring in Bovine α(s1)-Casein Variants by Peptide Microarray Immunoassay.

    Science.gov (United States)

    Lisson, Maria; Erhardt, Georg

    2016-01-01

    Immunoglobulin E epitope mapping of milk proteins reveals important information about their immunologic properties. Genetic variants of αS1-casein, one of the major allergens in bovine milk, are until now not considered when discussing the allergenic potential. Here we describe the complete procedure to assess the allergenicity of αS1-casein variants B and C, which are frequent in most breeds, starting from milk with identification and purification of casein variants by isoelectric focusing (IEF) and anion-exchange chromatography, followed by in vitro gastrointestinal digestion of the casein variants, identification of the resulting peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), in silico analysis of the variant-specific peptides as allergenic epitopes, and determination of their IgE-binding properties by microarray immunoassay with cow's milk allergic human sera.

  16. Epitope-dependent mechanisms of CD27 neutralization revealed by X-ray crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Obmolova, Galina; Teplyakov, Alexey; Malia, Thomas J.; Wunderler, Nicole; Kwok, Deborah; Barone, Linda; Sweet, Raymond; Ort, Tatiana; Scully, Michael; Gilliland, Gary L. (Janssen)

    2017-03-01

    CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different. The pharmacokinetics profiles could be epitope dependent. To identify the epitopes, we determined the crystal structure of the ternary complex between CD27 and the Fab fragments of these non-competing antibodies. The structure reveals the binding modes of the antibodies suggesting that their mechanisms of action are distinctly different and provides a possible explanation of the in vivo data.

  17. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules...

  18. Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18.

    Science.gov (United States)

    Baidya, Sunanda; Das, Rasel; Kabir, Md Golam; Arifuzzaman, Md

    2017-01-01

    Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through "reverse vaccinology" approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV.

  19. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  20. Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production.

    Science.gov (United States)

    Oyarzún, Patricio; Kobe, Bostjan

    2016-03-03

    Novel vaccination approaches based on rational design of B- and T-cell epitopes - epitope-based vaccines - are making progress in the clinical trial pipeline. The epitope-focused recombinant protein-based malaria vaccine (termed RTS,S) is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease. Progress made on methods such as recombinant DNA technology, advanced cell-culture techniques, immunoinformatics and rational design of immunogens are driving the development of these novel concepts. Synthetic recombinant proteins comprising both B- and T-cell epitopes can be efficiently produced through modern biotechnology and bioprocessing methods, and can enable the induction of large repertoires of immune specificities. In particular, the inclusion of appropriate CD4+ T-cell epitopes is increasingly considered a key vaccine component to elicit robust immune responses, as suggested by results coming from HIV-1 clinical trials. In silico strategies for vaccine design are under active development to address genetic variation in pathogens and several broadly protective "universal" influenza and HIV-1 vaccines are currently at different stages of clinical trials. Other methods focus on improving population coverage in target populations by rationally considering specificity and prevalence of the HLA proteins, though a proof-of-concept in humans has not been demonstrated yet. Overall, we expect immunoinformatics and bioprocessing methods to become a central part of the next-generation epitope-based vaccine development and production process.

  1. Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    S Pauliina Turunen

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

  2. Specific immunotherapy modifies allergen-specific CD4+ T cell responses in an epitope-dependent manner

    Science.gov (United States)

    Wambre, Erik; DeLong, Jonathan H.; James, Eddie A.; Torres-Chinn, Nadia; Pfützner, Wolfgang; Möbs, Christian; Durham, Stephen R.; Till, Stephen J.; Robinson, David; Kwok, William W.

    2014-01-01

    Background Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T cell epitope level is critical for the design of new allergy vaccine strategies. Objective To characterize allergen-specific T cell responses linked with allergy or peripheral tolerance and to determine how CD4+ T cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy (ASIT). Methods Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy and phenotype of the DR04:01-restricted TGP-specific T cell responses in ten grass pollen allergic, five non-atopic and six allergy vaccine-treated individuals was determined using an ex vivo pMHCII-tetramer approach. Results CD4+ T cells in allergic individuals are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. ASIT was associated with preferential deletion of allergen-specific TH2 cells and without significant change in frequency of TH1/TR1 cells. Conclusions Preferential allergen-specific TH2-cells deletion after repeated high doses antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy. PMID:24373351

  3. Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Jinfeng Ti

    Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

  4. Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation.

    Directory of Open Access Journals (Sweden)

    Angelo Duarte

    Full Text Available Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Type II collagen C2C epitope in human synovial fluid and serum after knee injury

    DEFF Research Database (Denmark)

    Kumahashi, N; Swärd, P; Larsson, S

    2015-01-01

    PURPOSE: Investigate in a cross-sectional study time-dependent changes of synovial fluid type II collagen epitope C2C concentrations after knee injury and correlate to other joint injury biomarkers. METHODS: Synovial fluid samples were aspirated between 0 days and 7 years after injury (n = 235...... = 0.403, P type II collagen (r = 0.444, P = 0.003), ARGS-aggrecan (r = 0.337, P ... with an immediate and sustained local degradation of type II collagen....

  7. Exposed and hidden lectin-binding epitopes at the surface of Borrelia burgdorferi

    Czech Academy of Sciences Publication Activity Database

    Stoitsova, S. R.; Grubhoffer, Libor; Nebesářová, Jana

    2003-01-01

    Roč. 48, č. 5 (2003), s. 654-658 ISSN 0015-5632 R&D Projects: GA AV ČR IAA6022001 Grant - others:National Research Council at the Ministry of Education and Science(BG) K-709/97 Institutional research plan: CEZ:AV0Z6022909 Keywords : Borrelia burgdorferi * lectin-binding epitopes Subject RIV: EE - Microbiology, Virology Impact factor: 0.857, year: 2003

  8. Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

    Science.gov (United States)

    Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

    2015-01-01

    Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

  9. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

    Directory of Open Access Journals (Sweden)

    George Janssen

    Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

  10. Expression of human apolipoprotein A-I epitopes in high density lipoproteins and in serum

    International Nuclear Information System (INIS)

    Marcel, Y.L.; Jewer, D.; Vezina, C.; Milthorp, P.; Weech, P.K.

    1987-01-01

    The expression and immunoreactivity of apolipoprotein (apo) A-I epitopes in high density lipoproteins (HDL) and serum has been investigated using two series of monoclonal antibodies (Mabs) which have been described elsewhere. Series 1 Mabs, identified as 3D4, 6B8, and 5G6, were obtained by immunization and screening with apoA-I, and series 2 Mabs, identified as 2F1, 4H1, 3G10, 4F7, and 5F6, were obtained by immunization and screening with HDL. These Mabs were characterized with respect to their binding to HDL particles in solution. In series 2 Mabs, 2F1, 3G10, and 4F7, which react with apoA-I CNBr-fragments 1 and 2, could precipitate 100% of 125 I-labeled HDL, while 4H1 and 5F6, which react with CNBr fragments 1 and 3, precipitated 90 and 60% of 125 I-labeled HDL, respectively. Therefore, three distinct epitopes mapped to CNBr fragments 1 and 2 have been identified which are expressed on all HDL particles, indicating that several antigenic do mains exist on apoA-I which have the same conformation on all apoA-I-containing lipoproteins. The Mabs reacting at these sites have significantly higher affinity constants for 125 I-labeled HDL than those that failed to precipitate 100% of HDL. This suggests that the high affinity Mabs react with apoA-I epitopes that are both expressed on all lipoproteins and located in thermo-dynamically stable regions of the molecules. All Mabs from series 1 precipitated 35% or less of 125 I-labeled HDL prepared from freshly collected serum, but the proportion of HDL particles expressing the epitopes for these Mabs doubled or more upon serum storage at 4 degrees C. The time course of the alteration of apoA-I antigen in vitro was measured in three normolipemic donors

  11. Future of an “Asymptomatic” T-cell Epitope-Based Therapeutic Herpes Simplex Vaccine

    Science.gov (United States)

    Dervillez, Xavier; Gottimukkala, Chetan; Kabbara, Khaled W.; Nguyen, Chelsea; Badakhshan, Tina; Kim, Sarah M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2012-01-01

    Summary Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as “asymptomatic” protective epitopes”) could boost local and systemic “natural” protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging “asymptomatic” T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease. PMID:22701511

  12. Flow shear stress differentially regulates endothelial uptake of nanocarriers targeted to distinct epitopes of PECAM-1.

    Science.gov (United States)

    Han, Jingyan; Shuvaev, Vladimir V; Davies, Peter F; Eckmann, David M; Muro, Silvia; Muzykantov, Vladimir R

    2015-07-28

    Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1-8dyn/cm(2), typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Determination of critical epitope of PcMab-47 against human podocalyxin

    Directory of Open Access Journals (Sweden)

    Shunsuke Itai

    2018-07-01

    Full Text Available Podocalyxin (PODXL is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA, flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

  14. Determination of critical epitope of PcMab-47 against human podocalyxin.

    Science.gov (United States)

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

    2018-07-01

    Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

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

    Science.gov (United States)

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

    2016-10-15

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

  16. Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

    Science.gov (United States)

    Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

    2014-01-25

    One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Preparation and application of epitope magnetic molecularly imprinted polymers for enrichment of sulfonamide antibiotics in water.

    Science.gov (United States)

    Hu, Yufeng; Wang, Cheng; Li, Xiangdao; Liu, Lifen

    2017-10-01

    Sulfonamides, which are widely used synthetic antibiotics, are hydrophilic and stable. They can easily migrate into the environment and aquatic animals, and increase the risk of cancer, drug resistance, and allergic symptoms if consumed by humans. Here, we developed an epitope magnetic imprinting approach to enrich multiple sulfonamide antibiotics from a water sample. Epitope magnetic molecularly imprinted polymers (EMMIPs) were prepared by free-radical polymerization using vinyl-functioned Fe 3 O 4 as a core, sulfanilamide (SA) as a dummy template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker. The performance of the EMMIPs was first evaluated by rebinding SA, and then an adsorption experiment was conducted to assess the extraction of multiple sulfonamide antibiotics containing the SA group. The binding experiments showed that the EMMIPs reached adsorption equilibrium in only 5 min with adsorption of SA at 2040 μg/g, compared with just 462 μg/g for the epitope magnetic non-imprinted polymers. EMMIPs were combined with HPLC for the detection of six sulfonamide antibiotics in surface water samples. The recoveries ranged from 79.3 to 92.4% and the relative standard deviations from 0.9 to 7.3%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting.

    Directory of Open Access Journals (Sweden)

    Ann-Marie Chacko

    Full Text Available Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1 facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.

  19. Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2016-12-01

    Full Text Available The coronavirus membrane (M protein acts as a dominant immunogen and is a major player in virus assembly. In this study, we prepared two monoclonal antibodies (mAbs; 1C3 and 4C7 directed against the transmissible gastroenteritis virus (TGEV M protein. The 1C3 and 4C7 mAbs both reacted with the native TGEV M protein in western blotting and immunofluorescence (IFA assays. Two linear epitopes, 243YSTEART249 (1C3 and 243YSTEARTDNLSEQEKLLHMV262 (4C7, were identified in the endodomain of the TGEV M protein. The 1C3 mAb can be used for the detection of the TGEV M protein in different assays. An IFA method for the detection of TGEV M protein was optimized using mAb 1C3. Furthermore, the ability of the epitope identified in this study to stimulate antibody production was also evaluated. An immunodominant epitope in the TGEV membrane protein endodomain was identified. The results of this study have implications for further research on TGEV replication.

  20. Technical presentation

    CERN Document Server

    FP Department

    2009-01-01

    07 April 2009 Technical presentation by Leuze Electronics: 14.00 – 15.00, Main Building, Room 61-1-017 (Room A) Photoelectric sensors, data identification and transmission systems, image processing systems. We at Leuze Electronics are "the sensor people": we have been specialising in optoelectronic sensors and safety technology for accident prevention for over 40 years. Our dedicated staff are all highly customer oriented. Customers of Leuze Electronics can always rely on one thing – on us! •\tFounded in 1963 •\t740 employees •\t115 MEUR turnover •\t20 subsidiaries •\t3 production facilities in southern Germany Product groups: •\tPhotoelectric sensors •\tIdentification and measurements •\tSafety devices

  1. Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities.

    Science.gov (United States)

    Starchenka, S; Bell, A J; Mwange, J; Skinner, M A; Heath, M D

    2017-01-01

    Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass. HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model. Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract. The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an 'immunodominant' Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept

  2. Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines.

    Science.gov (United States)

    Roukaerts, Inge D M; Grant, Chris K; Theuns, Sebastiaan; Christiaens, Isaura; Acar, Delphine D; Van Bockstael, Sebastiaan; Desmarets, Lowiese M B; Nauwynck, Hans J

    2017-01-02

    Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission. Copyright © 2016. Published by Elsevier B.V.

  3. Response of ELA-A1 horses immunized with lipopeptide containing an equine infectious anemia virus ELA-A1-restricted CTL epitope to virus challenge.

    Science.gov (United States)

    Ridgely, Sherritta L; Zhang, Baoshan; McGuire, Travis C

    2003-01-17

    Lipopeptide containing an ELA-A1-restricted cytotoxic T lymphocyte (CTL) epitope from the envelope surface unit (SU) protein of the EIAV(WSU5) strain was used to immunize three horses having the ELA-A1 haplotype. Peptide-specific ELA-A1-restricted CTL were induced in all three horses, although these were present transiently in PBMC. These horses were further immunized with lipopeptide containing the corresponding CTL epitope from the EIAV(PV) strain. Then, the three immunized horses and three non-immunized horses were challenged by intravenous inoculation with 300 TCID(50) EIAV(PV). All horses developed cell free viremia, fever and thrombocytopenia. However, there was a statistically lower fever and thrombocytopenia severity score in the immunized group. Shorter duration of plasma viral load in two of the three immunized horses likely explains the less severe clinical disease in this group. Results indicate that lipopeptide immunization had a protective effect against development of clinical disease following virus challenge.

  4. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Furebring Christina

    2009-03-01

    Full Text Available Abstract Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS blocks the Complement fragment C5a receptor (C5aR and formylated peptide receptor (FPR and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies

  5. Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

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    Anitra L Farrow

    2014-08-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5 vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83. This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes

  6. An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K

    2005-01-01

    Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have al.......The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php....

  7. Diversity of Francisella tularensis Schu4 antigens recognized by T lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine

    DEFF Research Database (Denmark)

    McMurry, Julie A; Gregory, Stephen H; Moise, Leonard

    2007-01-01

    The T lymphocyte antigens, which may have a role in protection against tularemia, were predicted by immunoinformatics analysis of Francisella tularensis Schu4. Twenty-seven class II putative promiscuous epitopes and 125 putative class I supertype epitopes were chosen for synthesis; peptides were...... responded to pools of 25 A2, A24, and B7 peptides, respectively. These data can aid in the development of novel epitope-based and subunit tularemia vaccines....

  8. Heat shock protein-derived T-cell epitopes contribute to autoimmune inflammation in pediatric Crohn's disease.

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    Gisella L Puga Yung

    Full Text Available Pediatric Crohn's disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohn's disease (CD is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues. The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF< and IFN induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.

  9. StreptInCor: a candidate vaccine epitope against S. pyogenes infections induces protection in outbred mice.

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    Edilberto Postol

    Full Text Available Infection with Streptococcus pyogenes (S. pyogenes can result in several diseases, particularly in children. S. pyogenes M protein is the major virulence factor, and certain regions of its N-terminus can trigger autoimmune sequelae such as rheumatic fever in susceptible individuals with untreated group A streptococcal pharyngitis. In a previous study, we utilized a large panel of human peripheral blood cells to define the C-terminal protective epitope StreptInCor (medical identity, which does not induce autoimmune reactions. We recently confirmed the results in HLA-transgenic mice. In the present study, we extended the experimental assays to outbred animals (Swiss mice. Herein, we demonstrate high titers of StreptInCor-specific antibodies, as well as appropriate T-cell immune responses. No cross-reaction to cardiac myosin was detected. Additionally, immunized Swiss mice exhibited 87% survival one month after challenge with S. pyogenes. In conclusion, the data presented herein reinforce previous results in humans and animals and further emphasize that StreptInCor could be an effective and safe vaccine for the prevention of S. pyogenes infections.

  10. Immunologic properties and therapeutic efficacy of a multivalent epitope-based vaccine against four Helicobacter pylori adhesins (urease, Lpp20, HpaA, and CagL) in Mongolian gerbils.

    Science.gov (United States)

    Guo, Le; Yin, Runting; Xu, Guangxian; Gong, Xiaojuan; Chang, Zisong; Hong, Dantong; Liu, Hongpeng; Ding, Shuqin; Han, Xuebo; Li, Yuan; Tang, Feng; Liu, Kunmei

    2017-12-01

    Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and adhesins, which are on the surface of H. pylori, play a pivotal role in binding to human gastric mucosa. In the present study, we constructed a multivalent epitope-based vaccine named CFAdE with seven carefully selected antigenic fragments from four H. pylori adhesins (urease, Lpp20, HpaA and CagL). The specificity, immunogenicity and ability to produce neutralizing antibodies of CFAdE were evaluated in BALB/c mice. After that, its therapeutic efficacy and protective immune mechanisms were explored in H. pylori-infected Mongolian gerbils. The results indicated that CFAdE could induce comparatively high levels of specific antibodies against urease, Lpp20, HpaA and CagL. Additionally, oral therapeutic immunization with CFAdE plus polysaccharide adjuvant (PA) significantly decreased H. pylori colonization compared with oral immunization with urease plus PA, and the protection was correlated with IgG and sIgA antibody and antigen-specific CD4 + T cells. This study indicated that the multivalent epitope-based vaccine, which targeted multiple adhesins in adherence of H. pylori to the gastric mucosa, is more effective than the univalent vaccine targeting urease only. This multivalent epitope-based vaccine may be a promising therapeutic candidate vaccine against H. pylori infection. © 2017 John Wiley & Sons Ltd.

  11. Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors

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    Zajac Paul

    2006-11-01

    Full Text Available Abstract Human polyomavirus BK (BKV has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351–450 and LTag533–626 by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.

  12. Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity.

    Science.gov (United States)

    Däumer, Martin P; Schneider, Beate; Giesen, Doris M; Aziz, Sheriff; Kaiser, Rolf; Kupfer, Bernd; Schneweis, Karl E; Schneider-Mergener, Jens; Reineke, Ulrich; Matz, Bertfried; Eis-Hübinger, Anna M

    2011-05-01

    Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

  13. Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products

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    Chen, F.; Haber, E.; Matsueda, G. R.

    1992-01-01

    The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

  14. Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection.

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    Hui-Jie Yang

    Full Text Available Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC, which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine.

  15. Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

    Science.gov (United States)

    Oscherwitz, Jon; Cease, Kemp B

    2015-01-01

    The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha

  16. Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

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    Jon Oscherwitz

    Full Text Available The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing

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

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    Jennifer Papuchon

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

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Usefulness of the nonself-self algorithm of HLA epitope immunogenicity in the specificity analysis of monospecific antibodies induced during pregnancy

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    Rene J Duquesnoy

    2015-05-01

    Full Text Available Background HLAMatchmaker is a program to analyze the epitope specificities of HLA antibodies. It considers each HLA allele as a string of eplets. Intralocus and interlocus comparisons between donor and recipient alleles offer a structural assessment of compatibility and an analysis of allele panel reactivity patterns can generate information about epitope specificities of HLA antibodies. However, HLAMatchmaker cannot always generate conclusive interpretations of reactivity patterns of all monospecific antibodies which by definition recognize single epitopes. Hypothesis We have therefore developed a new antibody analysis approach that utilizes the nonself-self algorithm of HLA epitope immunogenicity. It is based in the concept that HLA antibodies originate from B-cells with immunoglobulin receptors to self HLA epitopes on one given allele and which can be activated by epitopes defined by a few nonself residue differences whereas the remainder of the structural epitope of the immunizing allele consists of self residues. Methods Three human monoclonal class I antibodies from HLA typed women sensitized during pregnancy were tested in Ig-binding assays with single alleles on a Luminex platformFindings Three new HLA epitopes were identified; they are defined by combinations of nonself and self residues for one allele of the antibody producer. Conclusion The nonself-self paradigm of HLA epitope immunogenicity offers a second approach to analyze HLA antibody specificities.

  20. Alpha-gliadin genes from the A, B, and D genomes of wheat contain different sets of celiac disease epitopes

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    van Veelen Peter A

    2006-01-01

    Full Text Available Abstract Background Bread wheat (Triticum aestivum is an important staple food. However, wheat gluten proteins cause celiac disease (CD in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease. Results We obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87% contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome, as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific. Conclusion Our analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic

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

    Science.gov (United States)

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

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

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

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    Veronika I Zarnitsyna

    2016-06-01

    Full Text Available 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.

  3. Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

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    Pinghua Li

    Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

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

    Science.gov (United States)

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

    2012-01-01

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

  5. A Highly Conserved GEQYQQLR Epitope Has Been Identified in the Nucleoprotein of Ebola Virus by Using an In Silico Approach

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    Mohammad Tuhin Ali

    2015-01-01

    Full Text Available Ebola virus (EBOV is a deadly virus that has caused several fatal outbreaks. Recently it caused another outbreak and resulted in thousands afflicted cases. Effective and approved vaccine or therapeutic treatment against this virus is still absent. In this study, we aimed to predict B-cell epitopes from several EBOV encoded proteins which may aid in developing new antibody-based therapeutics or viral antigen detection method against this virus. Multiple sequence alignment (MSA was performed for the identification of conserved region among glycoprotein (GP, nucleoprotein (NP, and viral structural proteins (VP40, VP35, and VP24 of EBOV. Next, different consensus immunogenic and conserved sites were predicted from the conserved region(s using various computational tools which are available in Immune Epitope Database (IEDB. Among GP, NP, VP40, VP35, and VP30 protein, only NP gave a 100% conserved GEQYQQLR B-cell epitope that fulfills the ideal features of an effective B-cell epitope and could lead a way in the milieu of Ebola treatment. However, successful in vivo and in vitro studies are prerequisite to determine the actual potency of our predicted epitope and establishing it as a preventing medication against all the fatal strains of EBOV.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Expression and Stability of Foreign Epitopes Introduced into 3A Nonstructural Protein of Foot-and-Mouth Disease Virus

    Science.gov (United States)

    Li, Pinghua; Bai, Xingwen; Cao, Yimei; Han, Chenghao; Lu, Zengjun; Sun, Pu; Yin, Hong; Liu, Zaixin

    2012-01-01

    Foot-and-mouth disease virus (FMDV) is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa) HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags. PMID:22848509

  8. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    Science.gov (United States)

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-02-01

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

  10. Modelling a 3D structure for EgDf1 from shape Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand

    Science.gov (United States)

    Paulino, M.; Esteves, A.; Vega, M.; Tabares, G.; Ehrlich, R.; Tapia, O.

    1998-07-01

    EgDf1 is a developmentally regulated protein from the parasite Echinococcus granulosus related to a family of hydrophobic ligand binding proteins. This protein could play a crucial role during the parasite life cycle development since this organism is unable to synthetize most of their own lipids de novo. Furthermore, it has been shown that two related protein from other parasitic platyhelminths (Fh15 from Fasciola hepatica and Sm14 from Schistosoma mansoni) are able to confer protective inmunity against experimental infection in animal models. A three-dimensional structure would help establishing structure/function relationships on a knowledge based manner. 3D structures for EgDf1 protein were modelled by using myelin P2 (mP2) and intestine fatty acid binding protein (I-FABP) as templates. Molecular dynamics techniques were used to validate the models. Template mP2 yielded the best 3D structure for EgDf1. Palmitic and oleic acids were docked inside EgDf1. The present theoretical results suggest definite location in the secondary structure of the epitopic regions, consensus phosphorylation motifs and oleic acid as a good ligand candidate to EgDf1. This protein might well be involved in the process of supplying hydrophobic metabolites for membrane biosynthesis and for signaling pathways.

  11. Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an In Silico Approach

    Directory of Open Access Journals (Sweden)

    Idrees Muhammad

    2011-02-01

    Full Text Available Abstract Background HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An in silico approach was applied for the analysis of entire HCV proteome of Pakistani origin, aimed to identify the viral epitopes and their conservancy in HCV genotypes 1, 2 and 3 of diverse origin. Results Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide. Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100% conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2. Conclusions The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was

  12. Unusual antigen presentation offers new insight into HIV vaccine design.

    Science.gov (United States)

    McMichael, Andrew J; Picker, Louis J

    2017-06-01

    Recent findings with a rhesus monkey cytomegalovirus based simian immunodeficiency virus vaccine have identified strong CD8+ T cell responses that are restricted by MHC-E. Also mycobacteria specific CD8+ T cells, that are MHC-E restricted, have been identified. MHC-E therefore can present a wide range of epitope peptides to CD8+ T cells, alongside its well defined role in presenting a conserved MHC-class I signal peptide to the NKG2A/C-CD94 receptor on natural killer cells. Here we explore the antigen processing pathways involved in these atypical T cell responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization.

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    Anna D Koromyslova

    2017-11-01

    Full Text Available Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42 were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14, allosteric interference (Nano-32, and violation of normal capsid morphology (Nano-26 and Nano-85. Finally, we showed that two Nanobodies (Nano-26 and Nano-85 not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great

  14. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

    Science.gov (United States)

    2017-01-01

    Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to

  15. Human immune responses to H. pylori HLA Class II epitopes identified by immunoinformatic methods.

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    Songhua Zhang

    Full Text Available H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.

  16. Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

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    Tetsuji Kamata

    Full Text Available Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.

  17. Design, construction and evaluation of multi-epitope antigens for diagnosis of Lyme disease.

    Science.gov (United States)

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

    2017-12-23

    Introduction and objective. Lyme disease (LD) is the most common vector-borne disease in the temperate zone of the Northern Hemisphere. Diagnosis of LD is mainly based on clinical symptoms supported with serology (detection of anti-Borrelia antibodies) and is often misdiagnosed in areas of endemicity. In this study, the chimeric proteins (A/C-2, A/C-4 and A/C-7.1) consisting of B-cell epitopes of outer surface proteins OspA and OspC from Borrelia genospecies prevalent in Eastern Slovakia, were designed, over-expressed in E. coli, and used to detect specific anti-Borrelia antibodies in serologically characterized sera from patients with Lyme-like symptoms to evaluate their diagnostic potential. Results showed that chimeras vary in their immuno-reactivity when tested with human sera. Compared with the results obtained from a two-tier test, the application of recombinant multi-epitope chimeric proteins as diagnosis antigens, produced fair agreement in the case of A/C-2 (0.20<κ<0.40) and good agreement (0.60<κ<0.80) when A/C-7.1 was used as capture antigen. Chimera A/C-4 were excluded from further study due to loss of reactivity with OspA-specific antibodies. The combination of specific B-cell epitopes from OspA and OspC proteins may improve the diagnostic accuracy of serologic assays, but further studies are required to address this hypothesis.

  18. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø

    1997-01-01

    . Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...... with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized...

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

    OpenAIRE

    L?pez-Matas, M. Angeles; Gallego, Mayte; Iraola, V?ctor; Robinson, Douglas; Carn?s, Jer?nimo

    2013-01-01

    Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtain...

  20. Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Worning, Peder

    2004-01-01

    Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus......, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates...

  1. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, B; Pallesen, L; Jensen, LB

    1997-01-01

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted....... Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  2. HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified from herpes simplex virus glycoprotein D

    DEFF Research Database (Denmark)

    Chentoufi, Aziz Alami; Zhang, Xiuli; Lamberth, Kasper

    2008-01-01

    Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell ...... following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.......Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell...

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

    Science.gov (United States)

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

    2013-04-15

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

  4. Computational screening of Six Antigens for potential MHC class II restricted epitopes and evaluating its CD4+ T-Cell Responsiveness against Visceral Leishmaniasis

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    Manas Ranjan

    2017-12-01

    Full Text Available Visceral leishmaniasis is one of the most neglected tropical diseases for which no vaccine exists. In spite of extensive efforts, no successful vaccine is available against this dreadful infectious disease. To support the vaccine development, immunoinformatics approach was applied to search for potential MHC-classII restricted epitopes that can activate the immune cells. Initially, a total of 37 epitopes derived from six, stage dependent over expressed antigens were predicted, which were presented by at least 26 diverse MHC class II alleles including: DRB10101, DRB10301, DRB10401, DRB10404, DRB10405, DRB10701, DRB10802, DRB10901, DRB11101, DRB11302, DRB11501, DRB30101, DRB40101, DRB50101, DPA10103-DPB10401, DPA10103-DPB10201, DPA10201-DPB10101, DPA10103-DPB10301_DPB10401, DPA10301-DPB10402, DPA10201-DPB105021, DQA10102-DQB10602, DQA10401-DQB10402, DQA10501-QB10201, DQA10501-DQB10301, DQA10301-DQB10302 and DQA10101-DQB10501. Based on the population coverage analysis and HLA cross presentation ability, six epitopes namely, FDLFLFSNGAVVWWG (P1, YPVYPFLASNAALLN (P2, VYPFLASNAALLNLI (P3, LALLIMLYALIATQF (P4, LIMLYALIATQFSDD (P5, IMLYALIATQFSDDA (P6 were selected for further analysis. Stimulation with synthetic peptide alone or as a cocktail triggered the intracellular IFN-γ production. Moreover, specific IgG class of antibodies was detected in the serum of active VL cases against P1, P4, P and P6 in order to evaluate peptide effect on humoral immune response. Additionally, most of the peptides, except P2, were found to be non-inducer of CD4+ IL-10 against both active VL as well as treated VL subjects. Peptide immunogenicity was validated in BALB/c mice immunized with cocktail of synthetic peptide emulsified in complete Freund’s adjuvant/incomplete Freund’s adjuvant. The immunized splenocytes induced strong spleen cell proliferation upon parasite re-stimulation. Furthermore, an increased IFN-γ, IL-12, IL-17 and IL-22 production augmented with

  5. Induction of protective immunity against Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines.

    Science.gov (United States)

    Song, Xiaokai; Ren, Zhe; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2015-06-04

    Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The nature and combination of subunits used in epitope-based Schistosoma japonicum vaccine formulations affect their efficacy

    Directory of Open Access Journals (Sweden)

    Liu Feng

    2010-11-01

    Full Text Available Abstract Background Schistosomiasis remains a major public health problem in endemic countries and is caused by infections with any one of three primary schistosome species. Although there are no vaccines available to date, this strategy appears feasible since natural immunity develops in individuals suffering from repeated infection during a lifetime. Since vaccinations resulting in both Th1- and Th2-type responses have been shown to contribute to protective immunity, a vaccine formulation with the capacity for stimulating multiple arms of the immune response will likely be the most effective. Previously we developed partially protective, single Th- and B cell-epitope-based peptide-DNA dual vaccines (PDDV (T3-PDDV and B3-PDDV, respectively capable of eliciting immune responses against the Schistosoma japonicum 22.6 kDa tegument antigen (Sj22.6 and a 62 kDa fragment of myosin (Sj62, respectively. Results In this study, we developed PDDV cocktails containing multiple epitopes of S. japonicum from Sj22.6, Sj62 and Sj97 antigens by predicting cytotoxic, helper, and B-cell epitopes, and evaluated vaccine potential in vivo. Results showed that mice immunized with a single-epitope PDDV elicited either Tc, Th, or B cell responses, respectively, and mice immunized with either the T3- or B3- single-epitope PDDV formulation were partially protected against infection. However, mice immunized with a multicomponent (3 PDDV components formulation elicited variable immune responses that were less immunoprotective than single-epitope PDDV formulations. Conclusions Our data show that combining these different antigens did not result in a more effective vaccine formulation when compared to each component administered individually, and further suggest that immune interference resulting from immunizations with antigenically distinct vaccine targets may be an important consideration in the development of multicomponent vaccine preparations.

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

    Science.gov (United States)

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

    2017-12-01

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

  8. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E

    2014-01-01

    of differentiation on HIV-1-specific CD8+ T-cell populations(n = 128) spanning 11 different epitope targets. RESULTS: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR...

  9. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  10. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation

    DEFF Research Database (Denmark)

    Yusim, K.; Kesmir, Can; Gaschen, B.

    2002-01-01

    The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequenc...

  11. Identification of CD8(+) T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Lelic, A.; Parsons, R.

    2010-01-01

    bioinformatics methods to predict WNV-specific CD8(+) T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8(+) T cell...

  12. Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

    DEFF Research Database (Denmark)

    Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

    2007-01-01

    The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent...

  13. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate

    DEFF Research Database (Denmark)

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other reg...

  14. Comparing HLA shared epitopes in French Caucasian patients with scleroderma.

    Directory of Open Access Journals (Sweden)

    Doua F Azzouz

    Full Text Available Although many studies have analyzed HLA allele frequencies in several ethnic groups in patients with scleroderma (SSc, none has been done in French Caucasian patients and none has evaluated which one of the common amino acid sequences, (67FLEDR(71, shared by HLA-DRB susceptibility alleles, or (71TRAELDT(77, shared by HLA-DQB1 susceptibility alleles in SSc, was the most important to develop the disease. HLA-DRB and DQB typing was performed for a total of 468 healthy controls and 282 patients with SSc allowing FLEDR and TRAELDT analyses. Results were stratified according to patient's clinical subtypes and autoantibody status. Moreover, standardized HLA-DRß1 and DRß5 reverse transcriptase Taqman PCR assays were developed to quantify ß1 and ß5 mRNA in 20 subjects with HLA-DRB1*15 and/or DRB1*11 haplotypes. FLEDR motif is highly associated with diffuse SSc (χ(2 = 28.4, p<10-6 and with anti-topoisomerase antibody (ATA production (χ(2 = 43.9, p<10-9 whereas TRAELDT association is weaker in both subgroups (χ(2 = 7.2, p = 0.027 and χ(2 = 14.6, p = 0.0007 respectively. Moreover, FLEDR motif- association among patients with diffuse SSc remains significant only in ATA subgroup. The risk to develop ATA positive SSc is higher with double dose FLEDR than single dose with respectively, adjusted standardised residuals of 5.1 and 2.6. The increase in FLEDR motif is mostly due to the higher frequency of HLA-DRB1*11 and DRB1*15 haplotypes. Furthermore, FLEDR is always carried by the most abundantly expressed ß chain: ß1 in HLA DRB1*11 haplotypes and ß5 in HLA-DRB1*15 haplotypes.In French Caucasian patients with SSc, FLEDR is the main presenting motif influencing ATA production in dcSSc. These results open a new field of potential therapeutic applications to interact with the FLEDR peptide binding groove and prevent ATA production, a hallmark of severity in SSc.

  15. Design of a Heterotetravalent Synthetic Allergen That Reflects Epitope Heterogeneity and IgE Antibody Variability to Study Mast Cell Degranulation

    Science.gov (United States)

    Handlogten, Michael W.; Kiziltepe, Tanyel; Bilgicer, Basar

    2013-01-01

    SYNOPSIS This study describes the design of a heterotetravalent allergen (HtTA) as a multi-component experimental system that enables an integrative approach to study mast cell degranulation. The HtTA design allows presentation of two distinct haptens, each with a valency of two, thereby better reflecting the complexity of natural allergens by displaying epitope heterogeneity and IgE antibody variability. Using the HtTA design, synthetic allergens HtTA-1 and HtTA-2 were synthesized to model a combination of epitope/IgE affinities. HtTA-1 presented DNP and dansyl haptens (Kd = 22 and 54 nM for IgEDNP and IgEdansyl respectively), and HtTA-2 presented dansyl and the weak affinity DNP-Pro haptens (Kd = 550 nM for IgEDNP). Both HtTAs effectively induced degranulation when mast cells were primed with both IgEDNP and IgEdansyl antibodies. Interestingly, tetravalent DNP-Pro or bivalent dansyl were insufficient in stimulating a degranulation response, illustrating the significance of valency, affinity, and synergy in allergen-IgE interactions. Importantly, maximum degranulation with both HtTA-1 and HtTA-2 was observed when only 50% of the mast cell-bound IgEs were hapten specific (25% IgEdansyl + 25% IgEDNP). Taken together, this study establishes the HtTA system as a physiologically relevant experimental model and demonstrates its utility in elucidating critical mechanisms of mast cell degranulation. PMID:23050868

  16. Fine-mapping of immunodominant linear B-cell epitopes of the Staphylococcus aureus SEB antigen using short overlapping peptides.

    Directory of Open Access Journals (Sweden)

    Zhuo Zhao

    Full Text Available Staphylococcal enterotoxin B (SEB is one of the most potent Staphylococcus aureus exotoxins (SEs. Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97-114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257 were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97-112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97-112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.

  17. HLArestrictor-a tool for patient-specific predictions of HLA restriction elements and optimal epitopes within peptides

    DEFF Research Database (Denmark)

    Larsen, Malene Erup; Kloverpris, H.; Stryhn, A.

    2011-01-01

    ://www.cbs.dtu.dk/services/HLArestrictor ), which is based on the highly versatile and accurate NetMHCpan predictor, which here has been optimized for the identification of both the MHC restriction element and the corresponding minimal epitope of a T cell response in a given individual. As input, it requires high-resolution (i.e., 4-digit) HLA...... HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal...

  18. Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

    OpenAIRE

    Narat, Mojca; Biček, Ajda; Vadnjal, Robert; Benčina, Dušan

    2004-01-01

    Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs) that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY) shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of...

  19. Superior control of HIV-1 replication by CD8+ T cells targeting conserved epitopes: implications for HIV vaccine design.

    Directory of Open Access Journals (Sweden)

    Pratima Kunwar

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

  20. Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

    DEFF Research Database (Denmark)

    Schønning, Kristian; Bolmstedt, A; Novotny, J

    1998-01-01

    An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation....