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Sample records for cell epitopes delivered

  1. Influence of flanking sequences on presentation efficiency of a CD8+ cytotoxic T-cell epitope delivered by parvovirus-like particles.

    PubMed

    Rueda, P; Morón, G; Sarraseca, J; Leclerc, C; Casal, J I

    2004-03-01

    We have previously developed an antigen-delivery system based on hybrid recombinant porcine parvovirus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 protein of PPV carrying a foreign epitope at its N terminus. In this study, different constructs were made containing a CD8(+) T-cell epitope of chicken ovalbumin (OVA) to analyse the influence of the sequence inserted into VP2 on the correct processing of VLPs by antigen-presenting cells. We analysed the presentation of the OVA epitope inserted without flanking sequences or with either different natural flanking sequences or with the natural flanking sequences of a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus nucleoprotein, and as a dimer with or without linker sequences. All constructs were studied in terms of level of expression, assembly of VLPs and ability to deliver the inserted epitope into the MHC I pathway. The presentation of the OVA epitope was considerably improved by insertion of short natural flanking sequences, which indicated the relevance of the flanking sequences on the processing of PPV-VLPs. Only PPV-VLPs carrying two copies of the OVA epitope linked by two glycines were able to be properly processed, suggesting that the introduction of flexible residues between the two consecutive OVA epitopes may be necessary for the correct presentation of these dimers by PPV-VLPs. These results provide information to improve the insertion of epitopes into PPV-VLPs to facilitate their processing and presentation by MHC class I molecules.

  2. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    M Honda; R Wang; W Kong; M Kanekiyo; Q Akahata; L Xu; K Matsuo; K Natarajan; H Robinson; et al.

    2011-12-31

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  3. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    Honda, M.; Robinson, H.; Wang, R.; Kong, W.-P.; Kanekiyo, M.; Akahata, W.; Xu, L.; Matsuo, K.; Natarajan, K.; Asher, T. E.; Price, D. A.; Douek, D. C.; Margulies, D. H.; Nabel, G. J.

    2009-08-15

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  4. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    PubMed

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  5. Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

    PubMed Central

    Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

    2016-01-01

    X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Potocnakova, Lenka

    2016-01-01

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

  8. The two-faced T cell epitope

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

  10. Relevant B Cell Epitopes in Allergic Disease

    PubMed Central

    Pomés, Anna

    2010-01-01

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

  11. Equivalent T cell epitope promiscuity in ecologically diverse human pathogens.

    PubMed

    Wiens, Kirsten E; Swaminathan, Harish; Copin, Richard; Lun, Desmond S; Ernst, Joel D

    2013-01-01

    The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26293607

  13. Equivalent T Cell Epitope Promiscuity in Ecologically Diverse Human Pathogens

    PubMed Central

    Wiens, Kirsten E.; Swaminathan, Harish; Copin, Richard; Lun, Desmond S.; Ernst, Joel D.

    2013-01-01

    Background The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens’ epitopes to bind diverse HLA alleles, referred to as an epitope’s binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. Methods We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. Results We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. Conclusions Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation. PMID:23951341

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

    PubMed

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

    2015-01-01

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

  15. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    NASA Astrophysics Data System (ADS)

    Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

    1994-06-01

    IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

  16. Computational prediction of B cell epitopes from antigen sequences.

    PubMed

    Gao, Jianzhao; Kurgan, Lukasz

    2014-01-01

    Computational identification of B-cell epitopes from antigen chains is a difficult and actively pursued research topic. Efforts towards the development of method for the prediction of linear epitopes span over the last three decades, while only recently several predictors of conformational epitopes were released. We review a comprehensive set of 13 recent approaches that predict linear and 4 methods that predict conformational B-cell epitopes from the antigen sequences. We introduce several databases of B-cell epitopes, since the availability of the corresponding data is at the heart of the development and validation of computational predictors. We also offer practical insights concerning the use and availability of these B-cell epitope predictors, and motivate and discuss feature research in this area.

  17. Oral immunization with recombinant Lactococcus lactis delivering a multi-epitope antigen CTB-UE attenuates Helicobacter pylori infection in mice.

    PubMed

    Li, Xinyang; Xing, Yingying; Guo, Le; Lv, Xiaobo; Song, Hui; Xi, Tao

    2014-10-01

    Urease is an essential virulence factor and colonization factor for Helicobacter pylori (H. pylori) and is considered as an excellent vaccine candidate antigen. However, conventional technologies for preparing an injectable vaccine require purification of the antigenic protein and preparation of an adjuvant. Lactococcus lactis NZ9000 (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. In previous study, we constructed a multi-epitope vaccine, designated CTB-UE, which is composed of the mucosal adjuvant cholera toxin B subunit (CTB), three Th cell epitopes and two B-cell epitopes from urease subunits. To develop a novel type of oral vaccine against H. pylori, genetically modified L. lactis strains were established to secrete this epitope vaccine extracellularly in this study. Oral prophylactic immunization with recombinant L. lactis significantly elicited humoral anti-urease antibody responses (P < 0.001) and reduced the gastric colonization of H. pylori from 7.14 ± 0.95 to 4.68 ± 0.98 log10 CFU g(-1) stomach. This L. lactis oral vaccine offers a promising vaccine candidate for the control of H. pylori infection.

  18. Recent advances in B-cell epitope prediction methods

    PubMed Central

    2010-01-01

    Identification of epitopes that invoke strong responses from B-cells is one of the key steps in designing effective vaccines against pathogens. Because experimental determination of epitopes is expensive in terms of cost, time, and effort involved, there is an urgent need for computational methods for reliable identification of B-cell epitopes. Although several computational tools for predicting B-cell epitopes have become available in recent years, the predictive performance of existing tools remains far from ideal. We review recent advances in computational methods for B-cell epitope prediction, identify some gaps in the current state of the art, and outline some promising directions for improving the reliability of such methods. PMID:21067544

  19. Building Classifier Ensembles for B-Cell Epitope Prediction

    PubMed Central

    EL-Manzalawy, Yasser; Honavar, Vasant

    2015-01-01

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

  20. Removing T-cell epitopes with computational protein design

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Krishna, Sri; Anderson, Karen S

    2016-01-01

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

  2. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6

    PubMed Central

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

    2016-01-01

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

  3. T-cell epitope prediction methods: an overview.

    PubMed

    Desai, Dattatraya V; Kulkarni-Kale, Urmila

    2014-01-01

    The scientific community is overwhelmed by the voluminous increase in the quantum of data on biological systems, including but not limited to the immune system. Consequently, immunoinformatics databases are continually being developed to accommodate this ever increasing data and analytical tools are continually being developed to analyze the same. Therefore, researchers are now equipped with numerous databases, analytical and prediction tools, in anticipation of better means of prevention of and therapeutic intervention in diseases of humans and other animals. Epitope is a part of an antigen, recognized either by B- or T-cells and/or molecules of the host immune system. Since only a few amino acid residues that comprise an epitope (instead of the whole protein) are sufficient to elicit an immune response, attempts are being made to identify or predict this critical stretch or patch of amino acid residues, i.e., T-cell epitopes and B-cell epitopes to be included in multiple-subunit vaccines. T-cell epitope prediction is a challenge owing to the high degree of MHC polymorphism and disparity in the volume of data on various steps encountered in the generation and presentation of T-cell epitopes in the living systems. Many algorithms/methods developed to predict T-cell epitopes and Web servers incorporating the same are available. These are based on approaches like considering amphipathicity profiles of proteins, sequence motifs, quantitative matrices (QM), artificial neural networks (ANN), support vector machines (SVM), quantitative structure activity relationship (QSAR) and molecular docking simulations, etc. This chapter aims to introduce the reader to the principle(s) underlying some of these methods/algorithms as well as procedural and practical aspects of using the same.

  4. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    PubMed

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

    2016-02-01

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

  5. Mast Cells Produce a Unique Chondroitin Sulfate Epitope

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1992-01-01

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

  7. Bcipep: A database of B-cell epitopes

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2017-05-02

    Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex task. Despite substantial effort, limited advancement has been achieved over the last decade in the accuracy of epitope prediction methods, especially for those that rely on the sequence of the antigen only. Here, we present BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/), a web server for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from solved 3D structures, and on a large collection of linear epitopes downloaded from the IEDB database. The method displays results in a user-friendly and informative way, both for computer-savvy and non-expert users. We believe that BepiPred-2.0 will be a valuable tool for the bioinformatics and immunology community. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

    Antonets, D V; Maksiutov, A Z

    2010-01-01

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

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

    PubMed

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

    2001-06-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

    Raghava, Gajendra P. S.

    2013-01-01

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

  13. Immunoregulatory T cell epitope peptides: the new frontier in allergy therapy

    PubMed Central

    Prickett, S R; Rolland, J M; O'Hehir, R E

    2015-01-01

    Allergen immunotherapy (AIT) has been practised since 1911 and remains the only therapy proven to modify the natural history of allergic diseases. Although efficacious in carefully selected individuals, the currently licensed whole allergen extracts retain the risk of IgE-mediated adverse events, including anaphylaxis and occasionally death. This together with the need for prolonged treatment regimens results in poor patient adherence. The central role of the T cell in orchestrating the immune response to allergen informs the choice of T cell targeted therapies for down-regulation of aberrant allergic responses. Carefully mapped short synthetic peptides that contain the dominant T cell epitopes of major allergens and bind to a diverse array of HLA class II alleles, can be delivered intradermally into non-inflamed skin to induce sustained clinical and immunological tolerance. The short peptides from allergenic proteins are unable to cross-link IgE and possess minimal inflammatory potential. Systematic progress has been made from in vitro human models of allergen T cell epitope-based peptide anergy in the early 1990s, through proof-of-concept murine allergy models and early human trials with longer peptides, to the current randomized, double-blind, placebo-controlled clinical trials with the potential new class of synthetic short immune-regulatory T cell epitope peptide therapies. Sustained efficacy with few adverse events is being reported for cat, house dust mite and grass pollen allergy after only a short course of treatment. Underlying immunological mechanisms remain to be fully delineated but anergy, deletion, immune deviation and Treg induction all seem contributory to successful outcomes, with changes in IgG4 apparently less important compared to conventional AIT. T cell epitope peptide therapy is promising a safe and effective new class of specific treatment for allergy, enabling wider application even for more severe allergic diseases. PMID:25900315

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

    PubMed Central

    MELROSE, JAMES; SMITH, SUSAN; GHOSH, PETER

    2000-01-01

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

  15. Immunolocalization of cell wall carbohydrate epitopes in seaweeds: presence of land plant epitopes in Fucus vesiculosus L. (Phaeophyceae).

    PubMed

    Raimundo, Sandra Cristina; Avci, Utku; Hopper, Christina; Pattathil, Sivakumar; Hahn, Michael G; Popper, Zoë A

    2016-02-01

    Land plant cell wall glycan epitopes are present in Fucus vesiculosus. RG-I/AG mAbs recognize distinct glycan epitopes in structurally different galactans, and 3-linked glucans are also present in the cell walls. Cell wall-directed monoclonal antibodies (mAbs) have given increased knowledge of fundamental land plant processes but are not extensively used to study seaweeds. We profiled the brown seaweed Fucus vesiculosus glycome employing 155 mAbs that recognize predominantly vascular plant cell wall glycan components. The resulting profile was used to inform in situ labeling studies. Several of the mAbs recognized and bound to epitopes present in different thallus parts of Fucus vesiculosus. Antibodies recognizing arabinogalactan epitopes were divided into four groups based on their immunolocalization patterns. Group 1 bound to the stipe, blade, and receptacles. Group 2 bound to the antheridia, oogonia and paraphyses. Group 3 recognized antheridia cell walls and Group 4 localized on the antheridia inner wall and oogonia mesochite. This study reveals that epitopes present in vascular plant cell walls are also present in brown seaweeds. Furthermore, the diverse in situ localization patterns of the RG-I/AG clade mAbs suggest that these mAbs likely detect distinct epitopes present in structurally different galactans. In addition, 3-linked glucans were also detected throughout the cell walls of the algal tissues, using the β-glucan-directed LAMP mAb. Our results give insights into cell wall evolution, and diversify the available tools for the study of brown seaweed cell walls.

  16. Identification and Phylogeny of the First T Cell Epitope Identified from a Human Gut Bacteroides Species.

    PubMed

    Perez-Muñoz, Maria Elisa; Joglekar, Payal; Shen, Yi-Ju; Shen, Yi-Ji; Chang, Kuan Y; Peterson, Daniel A

    2015-01-01

    Host T cell reactivity toward gut bacterial epitopes has been recognized as part of disease pathogenesis. However, the specificity of T cells that recognize this vast number of epitopes has not yet been well described. After colonizing a C57BL/6J germ-free mouse with the human gut symbiotic bacteria Bacteroides thetaiotaomicron, we isolated a T cell that recognized these bacteria in vitro. Using this T cell, we mapped the first known non-carbohydrate T cell epitope within the phylum Bacteroidetes. The T cell also reacted to two other additional Bacteroides species. We identified the peptide that stimulated the T cell by using a genetic approach. Genomic data from the epitope-positive and epitope-negative bacteria explain the cross-reactivity of the T cell to multiple species. This epitope degeneracy should shape our understanding of the T cell repertoire stimulated by the complex microbiome residing in the gastrointestinal tract in both healthy and disease states.

  17. FRED--a framework for T-cell epitope detection.

    PubMed

    Feldhahn, Magdalena; Dönnes, Pierre; Thiel, Philipp; Kohlbacher, Oliver

    2009-10-15

    Over the last decade, immunoinformatics has made significant progress. Computational approaches, in particular the prediction of T-cell epitopes using machine learning methods, are at the core of modern vaccine design. Large-scale analyses and the integration or comparison of different methods become increasingly important. We have developed FRED, an extendable, open source software framework for key tasks in immunoinformatics. In this, its first version, FRED offers easily accessible prediction methods for MHC binding and antigen processing as well as general infrastructure for the handling of antigen sequence data and epitopes. FRED is implemented in Python in a modular way and allows the integration of external methods. FRED is freely available for download at http://www-bs.informatik.uni-tuebingen.de/Software/FRED.

  18. T-cell epitope peptide vaccines.

    PubMed

    Elsawa, Sherine F; Rodeberg, David A; Celis, Esteban

    2004-10-01

    T-cell immunotherapy is a promising treatment option for cancer. The identification of tumor antigens that are recognized by the immune system has allowed for the generation of vaccines for various malignancies. Due to the ease of manufacturing and characterizating peptide-based vaccines they have been used to stimulate antitumor T-cells. This article will review the use of peptide-based vaccines for the treatment of cancer by inducing antitumor T-lymphocyte responses.

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

    Cancer.gov

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Wang, Hsin-Wei; Pai, Tun-Wen

    2014-01-01

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

  4. Peanut T-cell epitope discovery: Ara h 1.

    PubMed

    Ramesh, Manish; Yuenyongviwat, Araya; Konstantinou, George N; Lieberman, Jay; Pascal, Mariona; Masilamani, Madhan; Sampson, Hugh A

    2016-06-01

    Identification of potential T-cell epitopes in the peanut major allergens is essential for development of peptide-based immunotherapy. Traditional methods of T-cell epitope discovery use overlapping short peptides spanning the full length of the protein in T-cell proliferation assays. Because large proteins, such as Ara h 1, require a large number of peptides, this limits screening to a small number of allergic subject-derived T-cell lines. We sought to identify candidate peptides of Ara h 1 that display promiscuous binding to MHC class II and induce TH2 cytokine production by T cells. In silico MHC class II binding prediction was performed with NetMHCIIpan 2.0 (peptide length, 15; 1-mer offset) and the most abundant class II alleles in the North American population and with an in vitro MHC class II peptide reporter assay performed in parallel, which used synthetic 15-mer peptides offset by 5 mer spanning the protein. High-resolution MHC class II typing and a T-cell proliferation assay using preselected peptides were performed with PBMCs from 98 subjects with peanut allergy and 14 healthy control subjects. IL-4, IL-13, IL-5, IFN-γ, and TNF-α levels were measured in culture supernatants. Thirty-six Ara h 1 peptides were identified by using in silico predictions and MHC class II binding assays. In combination with T-cell proliferation and cytokines secreted in T-cell assays, we have identified 4 vaccine candidate Ara h 1 peptides. Preselection of peptides by using in silico and in vitro approaches in combination with conventional methods appears to be an effective strategy for identifying peanut T-cell peptide vaccine candidates. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. T-Cell Proliferation Assay: Determination of Immunodominant T-Cell Epitopes of Food Allergens.

    PubMed

    Masilamani, Madhan; Pascal, Mariona; Sampson, Hugh A

    2017-01-01

    Characterization of allergen-specific T cells is critical to understand their contribution to disease pathogenesis. The identification of immunodominant T-cell epitopes is crucial for development of T-cell-based vaccines. Peptide-specific T-cell proliferation studies are usually performed in a library of short synthetic peptides (15mer or 20mer) with 3 or 5 offset spanning the entire length of the allergen. T-cell peptide epitopes lack the primary and tertiary structure of the native protein to cross-link IgE, but retain the ability to stimulate T cells. The peptides sequences can also be obtained either by in silico approaches and in vitro binding assays. The efficacy of T-cell epitope-based peptide immunotherapy has been proven in certain allergies. The present methodology describes T-cell proliferation assays using whole blood sample from allergic subjects.

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

    PubMed

    Zhang, Xue Wu

    2013-08-01

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

  9. Fine mapping of canine parvovirus B cell epitopes.

    PubMed

    López de Turiso, J A; Cortés, E; Ranz, A; García, J; Sanz, A; Vela, C; Casal, J I

    1991-10-01

    In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.

  10. Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes.

    PubMed

    Woo, Wai-Ping; Doan, Tracy; Herd, Karen A; Netter, Hans-Jürgen; Tindle, Robert W

    2006-04-01

    Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

  11. Limitations of homology searching for identification of T-cell antigens with library derived mimicry epitopes.

    PubMed

    Hiemstra, H S; van Veelen, P A; Geluk, A; Schloot, N C; de Vries, R R; Ottenhoff, T H; Roep, B O; Drijfhout, J W

    1999-09-01

    Mimicry epitopes that are recognized by T-cells can be identified through screening of synthetic peptide libraries. We have shown that these mimicry epitopes share sequence similarity with the corresponding natural epitopes and that mimicry sequences can be used for the definition of protein derived T-cell epitopes from databases. This can be done by either homology searching or pattern searching. Here we discuss the advantages and disadvantages of homology searching as an alternative for the generally applicable recognition pattern approach. We show that only for part of the library derived mimicry epitopes, the degree of similarity to the natural epitope may be high enough for successful homology searching in small databases.

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

    PubMed

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

    2006-10-01

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

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

    PubMed Central

    Doytchinova, Irini A; Guan, Pingping; Flower, Darren R

    2006-01-01

    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. PMID:16533401

  14. Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

    PubMed Central

    Choung, Rok Seon; Marietta, Eric V.; Van Dyke, Carol T.; Brantner, Tricia L.; Rajasekaran, John; Pasricha, Pankaj J.; Wang, Tianhao; Bei, Kang; Krishna, Karthik; Krishnamurthy, Hari K.; Snyder, Melissa R.; Jayaraman, Vasanth; Murray, Joseph A.

    2016-01-01

    Background Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. Methods Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. Results Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. Conclusions These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis. PMID:26824466

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

    PubMed

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

    2014-12-01

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

  16. HIV-1 Epitope Variability Is Associated with T Cell Receptor Repertoire Instability and Breadth.

    PubMed

    Balamurugan, Arumugam; Claiborne, Deon; Ng, Hwee L; Yang, Otto O

    2017-08-15

    Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of <3,000 RNA copies/ml over 17 to 179 weeks. The composition of TCRs targeting each epitope varied considerably in stability over time, although clonal stability (Sorensen index) was not significantly time dependent within this interval. However, TCR stability inversely correlated with epitope variability in the Los Alamos HIV-1 Sequence Database, consistent with TCR evolution being driven by epitope variation. Finally, a robust inverse correlation of TCR breadth against each epitope versus epitope variability further suggested that this variability drives TCR repertoire diversification. In the context of studies demonstrating rapidly shifting HIV-1 sequences in vivo, our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier to effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here, we demonstrate that the clonal stability and number of clones for the CTL response against an epitope are inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV

  17. Multiplicity of virus-encoded helper T-cell epitopes expressed on FBL-3 tumor cells.

    PubMed Central

    Iwashiro, M; Kondo, T; Shimizu, T; Yamagishi, H; Takahashi, K; Matsubayashi, Y; Masuda, T; Otaka, A; Fujii, N; Ishimoto, A

    1993-01-01

    To identify retroviral antigenic determinants recognized by CD4+ T helper cells during tumor rejection, we established four noncytolytic, helper-type, CD4+ T-cell clones by limiting dilution cultures of mixed lymphocyte-tumor cultures from mice immune to a Friend virus-induced tumor, FBL-3. Among these, three T helper cell clones were isolated from C57BL/6 mice and the fourth was isolated from a (BALB/c x C57BL/6)F1 mouse. All these clones proliferated in response to the immunizing FBL-3 tumor cells in a major histocompatibility complex class II-restricted manner. Each clone expressed a distinct T-cell receptor with a characteristic combination of alpha and beta chains. The localization of helper T-cell determinants on viral proteins was analyzed with recombinant vaccinia viruses expressing Friend murine leukemia virus (F-MuLV) gag or env genes or shorter fragments of the env gene. Epitopes recognized by these T-cell clones were mapped to at least two distinct portions in the env region of the F-MuLV genome. These epitopes were identified more precisely with synthetic peptides derived from the F-MuLV envelope protein sequence. One of these epitopes was common to Friend and Moloney MuLVs and was located in the N-terminal region of the gp70 glycoprotein at amino acids 122 to 141. The second epitope, which was recognized in the context of hybrid I-Eb/d major histocompatibility complex class II molecule, was located close to the C-terminal end of gp70 at amino acids 462 to 479. In addition, a possible third epitope was located in the N-terminal half of the gp70 sequence and differed from the first epitope in that it was not cross-reactive with the Moloney MuLV envelope protein. PMID:7687300

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

    PubMed

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

    2016-02-01

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

  19. Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes

    PubMed Central

    Lindestam Arlehamn, Cecilia S.; Paul, Sinu; Mele, Federico; Huang, Charlie; Greenbaum, Jason A.; Vita, Randi; Sidney, John; Peters, Bjoern; Sallusto, Federica; Sette, Alessandro

    2015-01-01

    A previous unbiased genome-wide analysis of CD4 Mycobacterium tuberculosis (MTB) recognition using peripheral blood mononuclear cells from individuals with latent MTB infection (LTBI) or nonexposed healthy controls (HCs) revealed that certain MTB sequences were unexpectedly recognized by HCs. In the present study, it was found that, based on their pattern of reactivity, epitopes could be divided into LTBI-specific, mixed reactivity, and HC-specific categories. This pattern corresponded to sequence conservation in nontuberculous mycobacteria (NTMs), suggesting environmental exposure as an underlying cause of differential reactivity. LTBI-specific epitopes were found to be hyperconserved, as previously reported, whereas the opposite was true for NTM conserved epitopes, suggesting that intragenus conservation also influences host pathogen adaptation. The biological relevance of this observation was demonstrated further by several observations. First, the T cells elicited by MTB/NTM cross-reactive epitopes in HCs were found mainly in a CCR6+CXCR3+ memory subset, similar to findings in LTBI individuals. Thus, both MTB and NTM appear to elicit a phenotypically similar T-cell response. Second, T cells reactive to MTB/NTM-conserved epitopes responded to naturally processed epitopes from MTB and NTMs, whereas T cells reactive to MTB-specific epitopes responded only to MTB. Third, cross-reactivity could be translated to antigen recognition. Several MTB candidate vaccine antigens were cross-reactive, but others were MTB-specific. Finally, NTM-specific epitopes that elicit T cells that recognize NTMs but not MTB were identified. These epitopes can be used to characterize T-cell responses to NTMs, eliminating the confounding factor of MTB cross-recognition and providing insights into vaccine design and evaluation. PMID:25548174

  20. DM determines the cryptic and immunodominant fate of T cell epitopes.

    PubMed

    Nanda, N K; Sant, A J

    2000-09-18

    The ability of the immune system to focus T cell responses against a select number of potential epitopes of a complex antigen is termed immunodominance. Epitopes that trigger potent T cell activation, after in vivo priming, are classified as immunodominant. By contrast, determinants that fail to elicit any response are called cryptic. DM, a major histocompatibility complex (MHC) heterodimer, plays a pivotal role in the presentation of MHC class II-restricted epitopes by catalyzing the exchange of class II-associated invariant chain peptide with the antigen-derived peptides within the MHC class II binding groove. Using L cells transfected with genes for MHC class II, invariant chain, and DM, we have studied the contribution of DM in the presentation of two cryptic (peptide 11-25 and peptide 20-35) and one dominant (peptide 106-116) epitope of hen egg white lysozyme (HEL). Cells lacking DM heterodimers efficiently display the determinants HEL 11-25 and HEL 20-35 to T cells. Strikingly, however, cells expressing DM are severely compromised in their ability to present the cryptic HEL 11-25/A(d) and 20-35/A(d) epitopes. DM-mediated antagonism of HEL 11-25/A(d) and 20-35/A(d) presentation could thus be central to 11-25/A(d) and 20-35/A(d) being cryptic epitopes in the HEL system. Interestingly, the display of the immunodominant epitope of HEL, 106-116/E(d), and of a dominant epitope of sperm whale myoglobin (SWM), 102-118/A(d), is entirely dependent on the expression of DM. Thus, cells lacking DM molecules are unable to efficiently express HEL 106-116/E(d) and SWM 102-118/A(d) determinants. We conclude that the DM heterodimers direct the immunodominant and cryptic fate of antigenic epitopes in vivo.

  1. Dm Determines the Cryptic and Immunodominant Fate of T Cell Epitopes

    PubMed Central

    Nanda, Navreet K.; Sant, Andrea J.

    2000-01-01

    The ability of the immune system to focus T cell responses against a select number of potential epitopes of a complex antigen is termed immunodominance. Epitopes that trigger potent T cell activation, after in vivo priming, are classified as immunodominant. By contrast, determinants that fail to elicit any response are called cryptic. DM, a major histocompatibility complex (MHC) heterodimer, plays a pivotal role in the presentation of MHC class II–restricted epitopes by catalyzing the exchange of class II–associated invariant chain peptide with the antigen-derived peptides within the MHC class II binding groove. Using L cells transfected with genes for MHC class II, invariant chain, and DM, we have studied the contribution of DM in the presentation of two cryptic (peptide 11–25 and peptide 20–35) and one dominant (peptide 106–116) epitope of hen egg white lysozyme (HEL). Cells lacking DM heterodimers efficiently display the determinants HEL 11–25 and HEL 20–35 to T cells. Strikingly, however, cells expressing DM are severely compromised in their ability to present the cryptic HEL 11–25/Ad and 20–35/Ad epitopes. DM-mediated antagonism of HEL 11–25/Ad and 20–35/Ad presentation could thus be central to 11–25/Ad and 20–35/Ad being cryptic epitopes in the HEL system. Interestingly, the display of the immunodominant epitope of HEL, 106–116/Ed, and of a dominant epitope of sperm whale myoglobin (SWM), 102–118/Ad, is entirely dependent on the expression of DM. Thus, cells lacking DM molecules are unable to efficiently express HEL 106–116/Ed and SWM 102–118/Ad determinants. We conclude that the DM heterodimers direct the immunodominant and cryptic fate of antigenic epitopes in vivo. PMID:10993909

  2. Identification and Phylogeny of the First T Cell Epitope Identified from a Human Gut Bacteroides Species

    PubMed Central

    Perez-Muñoz, Maria Elisa; Joglekar, Payal; Shen, Yi-Ji; Chang, Kuan Y.; Peterson, Daniel A.

    2015-01-01

    Host T cell reactivity toward gut bacterial epitopes has been recognized as part of disease pathogenesis. However, the specificity of T cells that recognize this vast number of epitopes has not yet been well described. After colonizing a C57BL/6J germ-free mouse with the human gut symbiotic bacteria Bacteroides thetaiotaomicron, we isolated a T cell that recognized these bacteria in vitro. Using this T cell, we mapped the first known non-carbohydrate T cell epitope within the phylum Bacteroidetes. The T cell also reacted to two other additional Bacteroides species. We identified the peptide that stimulated the T cell by using a genetic approach. Genomic data from the epitope-positive and epitope-negative bacteria explain the cross-reactivity of the T cell to multiple species. This epitope degeneracy should shape our understanding of the T cell repertoire stimulated by the complex microbiome residing in the gastrointestinal tract in both healthy and disease states. PMID:26637014

  3. B-1 Cell Immunoglobulin Directed Against Oxidation-Specific Epitopes

    PubMed Central

    Tsiantoulas, Dimitrios; Gruber, Sabrina; Binder, Christoph J.

    2013-01-01

    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. PMID:23316200

  4. Mapping of the immunodominant T cell epitopes of the protein topoisomerase I

    PubMed Central

    Veeraraghavan, S; Renzoni, E; Jeal, H; Jones, M; Hammer, J; Wells, A; Black, C; Welsh, K; du Bois, R M

    2004-01-01

    Objectives: To identify the immunodominant T cell epitopes of the topoisomerase I protein in patients with systemic sclerosis (SSc) and control subjects, using computational analysis software (TEPITOPE) and T cell proliferation assays. Methods: Six oligopeptides, predicted by TEPITOPE software as potential topoisomerase protein epitopes, were used to perform T cell proliferation assays in 21 patients with SSc and 15 healthy controls. Results: A positive response to at least one of the peptides was seen in 10/21 patients and 7/15 healthy controls. Among responders, the proliferative response was limited to a single peptide in 6/7 healthy controls, whereas 5/10 patients responded to more than one peptide. In responding patients a significant correlation was found between disease duration and number of peptides inducing a response (p = 0.007). Conclusions: Several T cell epitopes of the topoisomerase I protein have been identified and evidence has been found to suggest epitope spreading in patients with SSc. PMID:15249326

  5. TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes

    PubMed Central

    Chowell, Diego; Krishna, Sri; Cocita, Clément; Shu, Jack; Tan, Xuefang; Greenberg, Philip D.; Klavinskis, Linda S.; Blattman, Joseph N.; Anderson, Karen S.

    2015-01-01

    Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8+ T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development. PMID:25831525

  6. A meta-learning approach for B-cell conformational epitope prediction.

    PubMed

    Hu, Yuh-Jyh; Lin, Shun-Chien; Lin, Yu-Lung; Lin, Kuan-Hui; You, Shun-Ning

    2014-11-18

    One of the major challenges in the field of vaccine design is identifying B-cell epitopes in continuously evolving viruses. Various tools have been developed to predict linear or conformational epitopes, each relying on different physicochemical properties and adopting distinct search strategies. We propose a meta-learning approach for epitope prediction based on stacked and cascade generalizations. Through meta learning, we expect a meta learner to be able integrate multiple prediction models, and outperform the single best-performing model. The objective of this study is twofold: (1) to analyze the complementary predictive strengths in different prediction tools, and (2) to introduce a generic computational model to exploit the synergy among various prediction tools. Our primary goal is not to develop any particular classifier for B-cell epitope prediction, but to advocate the feasibility of meta learning to epitope prediction. With the flexibility of meta learning, the researcher can construct various meta classification hierarchies that are applicable to epitope prediction in different protein domains. We developed the hierarchical meta-learning architectures based on stacked and cascade generalizations. The bottom level of the hierarchy consisted of four conformational and four linear epitope prediction tools that served as the base learners. To perform consistent and unbiased comparisons, we tested the meta-learning method on an independent set of antigen proteins that were not used previously to train the base epitope prediction tools. In addition, we conducted correlation and ablation studies of the base learners in the meta-learning model. Low correlation among the predictions of the base learners suggested that the eight base learners had complementary predictive capabilities. The ablation analysis indicated that the eight base learners differentially interacted and contributed to the final meta model. The results of the independent test demonstrated that

  7. CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor

    PubMed Central

    Ascough, Stephanie; Ingram, Rebecca J.; Chu, Karen K. Y.; Musson, Julie A.; Moore, Stephen J.; Gallagher, Theresa; Baillie, Les; Williamson, Ethel D.; Robinson, John H.; Maillere, Bernard; Boyton, Rosemary J.; Altmann, Daniel M.

    2016-01-01

    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 analyzed 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 characterized epitopes that elicited a response following immunization 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, as 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 transgenics. 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 influenced by the

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

    PubMed

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

    2016-09-01

    DNA-based vaccine is a promising candidate for immunization and induction of a T-cell-focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi-functional T response against multi-TB antigens, a multi-epitope DNA vaccine and a 'protein backbone grafting' design method is adopted to graft five discontinuous T-cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T-cell epitopes derived from ESAT-6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope-scaffold construct was significantly more potent to induce IFN-γ(+) T response to five T-cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi-functional-CD4(+) T response. It also enhanced granzyme B(+) CTL and IL-2(+) CD8(+) T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi-epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  9. Cell-mediated responses of immunized vervet monkeys to defined Leishmania T-cell epitopes.

    PubMed Central

    Curry, A J; Jardim, A; Olobo, J O; Olafson, R W

    1994-01-01

    A population of vervet monkeys was immunized with killed parasites and infected with Leishmania major promastigotes either by needle or by infected-fly bite. The responses of recovered monkeys to mitogens, killed parasites, and molecularly defined T-cell epitopes were then compared with those of control animals. Peripheral blood mononuclear cells (PBMC) from both naive and recovered animals proliferated strongly in response to both B- and T-cell mitogens, although the responses of the recovered animals were less strong than those of the naive animals. Cells from recovered vervets, but not those from naive vervets, also proliferated in response to parasite antigens and synthetic T-cell epitopes. Likewise, cells from recovered animals released gamma interferon and either interleukin 2 (IL-2) or IL-4 into culture media in response to both of the above-mentioned antigens, whereas cells from control animals did not. The fact that no IL-5 could be measured following parasite antigen or synthetic T-cell epitope stimulation of PBMC suggested that cells proliferating in response to these molecules belonged to the Th1 subset. Phenotypic analysis of the PBMC showed a marked increase in T-cell but not B-cell populations in recovered animals. Among this population was an increased number of CD45R0+ memory cells. The data from this study are in keeping with the earlier finding that vervet monkeys provide an excellent model system for leishmaniasis. Further, these data support the contention that synthetic T-cell epitopes are prime candidates for molecularly defined Leishmania vaccines. PMID:7513306

  10. Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA).

    PubMed

    O'Hara, Joanne M; Kasten-Jolly, Jane C; Reynolds, Claire E; Mantis, Nicholas J

    2014-01-01

    Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design.

  11. Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.

    PubMed

    Sharon, Jacqueline; Rynkiewicz, Michael J; Lu, Zhaohua; Yang, Chiou-Ying

    2014-05-01

    Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B-cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding can be localized to specific regions of antigens by immunochemical assays. The epitopes are then identified with mapping methods such as X-ray crystallography of antigen-antibody complexes, antibody inhibition of hydrogen-deuterium exchange in the antigen, antibody-induced alteration of the nuclear magnetic resonance spectrum of the antigen, and experimentally validated computational docking of antigen-antibody complexes. The diversity in shape, size and structure of protective B-cell epitopes, and the increasing importance of protective B-cell epitope discovery to development of vaccines and antibody therapeutics are illustrated through examples from different microbe categories, with emphasis on epitopes targeted by broadly neutralizing antibodies to pathogens of high antigenic variation. Examples include the V-shaped Ab52 glycan epitope in the O-antigen of Francisella tularensis, the concave CR6261 peptidic epitope in the haemagglutinin stem of influenza virus H1N1, and the convex/concave PG16 glycopeptidic epitope in the gp120 V1/V2 loop of HIV type 1.

  12. Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1.

    PubMed

    Asemissen, Anne Marie; Keilholz, Ulrich; Tenzer, Stefan; Müller, Margret; Walter, Steffen; Stevanovic, Stefan; Schild, Hansjörg; Letsch, Anne; Thiel, Eckhard; Rammensee, Hans-Georg; Scheibenbogen, Carmen

    2006-12-15

    The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy. Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC. One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer. In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

  13. Stretching cells and delivering drugs with bubbles

    NASA Astrophysics Data System (ADS)

    Ohl, Claus-Dieter; Li, Fenfang; Chon U, Chan; Gao, Yu; Xu, Chenjie

    2015-11-01

    In this talk we'll review our work on impulsive cell stretching using cavitation bubbles and magnetic microbubbles for drug delivery. For sufficient short times cells can sustain a much larger areal strain than the yield strain obtained from quasi-static stretching. Experiments with red blood cells show that even then the rupture of the cell is slow process; it is caused by diffusive swelling rather than mechanical violation of the plasma membrane. In the second part we'll discuss bubbles coated with magnetic and drug loaded particles. These bubbles offer an interesting vector for on demand delivery of drugs using mild ultrasound and magnetic fields. We report on basic experiments in microfluidic channels revealing the release of the agent during bubble oscillations and first in vivo validation with a mouse tumor model. Singapore National Research Foundations Competitive Research Program funding (NRF-CRP9-2011-04).

  14. In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model

    PubMed Central

    Gutiérrez, Andres H.; Loving, Crystal; Moise, Leonard; Terry, Frances E.; Brockmeier, Susan L.; Hughes, Holly R.; Martin, William D.; De Groot, Anne S.

    2016-01-01

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs. PMID:27411061

  15. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance

    PubMed Central

    van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H. J.; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines. PMID:27333291

  16. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

    PubMed

    Rosendahl Huber, Sietske K; Luimstra, Jolien J; van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H J; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

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

    PubMed

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

    2011-07-30

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

  18. Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

    PubMed Central

    Lesénéchal, Mylène; Becquart, Laurence; Lacoux, Xavier; Ladavière, Laurent; Baida, Renata C. P.; Paranhos-Baccalà, Glaucia; da Silveira, José Franco

    2005-01-01

    Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules. PMID:15699429

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

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  20. Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

    PubMed

    Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C

    2015-01-01

    The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

  1. Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

    SciTech Connect

    Yu Hua; Jiang Lifang . E-mail: jianglf909@yahoo.com.cn; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-03-15

    Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

  2. Cross‐reactivity of hepatitis C virus specific vaccine‐induced T cells at immunodominant epitopes

    PubMed Central

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul

    2014-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS31073 and NS31446, and multiple variants for epitope NS31406 that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross‐reactivity of vaccine‐induced T cells was determined using variant peptides in IFN‐γ ELISPOT assays. Vaccine‐induced T cells targeted approximately 90% of NS31073 genotype 1 sequences and 50% of NS31446 genotype 1 and 3 sequences. For NS31406, 62% of subtype‐1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS31406 that was maximally cross‐reactive. In vitro priming assays showed that of those tested the NS31406 vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross‐reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross‐reactive T cells at immunodominant epitopes. The degree of cross‐reactivity varies between epitopes and may be HCV‐subtype specific. PMID:25263407

  3. Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.

    PubMed

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3(1073) and NS3(1446), and multiple variants for epitope NS3(1406) that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross-reactivity of vaccine-induced T cells was determined using variant peptides in IFN-γ ELISPOT assays. Vaccine-induced T cells targeted approximately 90% of NS3(1073) genotype 1 sequences and 50% of NS3(1446) genotype 1 and 3 sequences. For NS3(1406), 62% of subtype-1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3(1406) that was maximally cross-reactive. In vitro priming assays showed that of those tested the NS3(1406) vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross-reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross-reactive T cells at immunodominant epitopes. The degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.

  4. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    PubMed

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. A novel immunization approach for dengue infection based on conserved T cell epitopes formulated in calcium phosphate nanoparticles.

    PubMed

    Huang, Xiaofang; Karabudak, Aykan; Comber, Joseph D; Philip, Mohan; Morcol, Tulin; Philip, Ramila

    2017-09-21

    Dengue virus (DV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Most DV vaccine approaches are focused on generating antibody mediated responses; one such DV vaccine is approved for use in humans but its efficacy is limited. While it is clear that T cell responses play important role in DV infection and subsequent disease manifestations, fewer studies are aimed at developing vaccines that induce robust T cells responses. Potent T cell based vaccines require two critical components: the identification of specific T cell stimulating MHC associated peptides, and an optimized vaccine delivery vehicle capable of simultaneously delivering the antigens and any required adjuvants. We have previously identified and characterized DV specific HLA-A2 and -A24 binding DV serotypes conserved epitopes, and the feasibility of an epitope based vaccine for DV infection. In this study, we build on those previous studies and describe an investigational DV vaccine utilizing T cell epitopes incorporated into a calcium phosphate nanoparticle (CaPNP) delivery system. This study presents a comprehensive analysis of functional immunogenicity of DV CaPNP/multipeptide formulations in vitro and in vivo and demonstrates the CaPNP/multipeptide vaccine is capable of inducing T cell responses against all four serotypes of DV. This synthetic vaccine is also cost effective, straightforward to manufacture, and stable at room temperature in a lyophilized form. This formulation may serve as an effective candidate DV vaccine that protects against all four serotypes as either a prophylactic or therapeutic vaccine.

  6. Enhanced immunogenicity of a functional enzyme by T cell epitope modification

    PubMed Central

    Mucha, Jeanette M; Stickler, Marcia M; Poulose, AJ; Ganshaw, Grant; Saldajeno, Mae; Collier, Kathy; Huang, Manley T; Harding, Fiona A

    2002-01-01

    Background T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. Results Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. Conclusions With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics. PMID:11869454

  7. Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

    PubMed

    Bozzacco, Leonia; Yu, Haiqiang; Dengjel, Jörn; Trumpfheller, Christine; Zebroski, Henry A; Zhang, Nawei; Küttner, Victoria; Ueberheide, Beatrix M; Deng, Haiteng; Chait, Brian T; Steinman, Ralph M; Mojsov, Svetlana; Fenyö, David

    2012-01-01

    Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

  8. T-cell epitope conservation across allergen species is a major determinant of immunogenicity

    PubMed Central

    Westernberg, Luise; Schulten, Véronique; Greenbaum, Jason A; Natali, Sara; Tripple, Victoria; McKinney, Denise M.; Frazier, April; Hofer, Heidi; Wallner, Michael; Sallusto, Federica; Sette, Alessandro; Peters, Bjoern

    2016-01-01

    Background Patients with pollen allergies are frequently poly-sensitized. Pollen contain epitopes that are conserved across multiple species. Objective Demostrate that cross-reactive T-cells which recognize conserved epitopes show higher levels of expansion than T-cells recognizing monospecific epitopes, due to more frequent stimulation. Method RNA was sequenced from nine pollens and the reads were assembled de-novo into >50,000 transcripts. T-cell epitopes from Timothy Grass (Phl p) were examined for conservation in these transcripts and this was correlated with their ability to induce T-cell responses. T-cells were expanded in vitro with Phl p-derived peptides and tested for cross-reactivity to pollen extracts in ELISPOT assays. Results We found that antigenic proteins are more conserved than non-immunogenic proteins in Phl p pollen. Additionally, Phl p epitopes that were highly conserved across pollens elicited more T-cell responses in grass allergic donors than less conserved ones. Moreover, conservation of a Phl p peptide at the transcriptomic level correlated with the ability of that peptide to trigger T-cells that were cross-reactive with other non-Phl p pollen extracts. Conclusion We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within Phl p as well as their ability to induce cross-reactive T-cell responses. T-cells recognizing conserved epitopes may be more prominent because they can be stimulated by a broader range of pollens and thereby drive poly-sensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of poly-sensitization and target multiple pollen allergies. PMID:26883464

  9. Newly Exerted T Cell Pressures on Mutated Epitopes following Transmission Help Maintain Consensus HIV-1 Sequences.

    PubMed

    Eriksson, Emily M; Liegler, Teri; Keh, Chris E; Karlsson, Annika C; Holditch, Sara J; Pilcher, Christopher D; Loeb, Lisa; Nixon, Douglas F; Hecht, Frederick M

    2014-01-01

    CD8+ T cells are important for HIV-1 virus control, but are also a major contributing factor that drives HIV-1 virus sequence evolution. Although HIV-1 cytotoxic T cell (CTL) escape mutations are a common aspect during HIV-1 infection, less is known about the importance of T cell pressure in reversing HIV-1 virus back to a consensus sequences. In this study we aimed to assess the frequency with which reversion of transmitted mutations in T cell epitopes were associated with T cell responses to the mutation. This study included 14 HIV-1 transmission pairs consisting of a 'source' (virus-donor) and a 'recipient' (newly infected individual). Non-consensus B sequence amino acids (mutations) in T cell epitopes in HIV-1 gag regions p17, p24, p2 and p7 were identified in each pair and transmission of mutations to the recipient was verified with population viral sequencing. Longitudinal analyses of the recipient's viral sequence were used to identify whether reversion of mutations back to the consensus B sequence occurred. Autologous 12-mer peptides overlapping by 11 were synthesized, representing the sequence region surrounding each reversion and longitudinal analysis of T cell responses to source-derived mutated and reverted epitopes were assessed. We demonstrated that mutations in the source were frequently transmitted to the new host and on an average 17 percent of mutated epitopes reverted to consensus sequence in the recipient. T cell responses to these mutated epitopes were detected in 7 of the 14 recipients in whom reversion occurred. Overall, these findings indicate that transmitted non-consensus B epitopes are frequently immunogenic in HLA-mismatched recipients and new T cell pressures to T cell escape mutations following transmission play a significant role in maintaining consensus HIV-1 sequences.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

    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

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

    PubMed

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

    2014-05-01

    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. Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  15. Conservation of HIV-1 T cell epitopes across time and clades: validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine.

    PubMed

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou; Ardito, Matthew T; Boyle, Christine M; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D; De Groot, Anne S

    2012-12-14

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine.

  16. Conservation of HIV-1 T cell epitopes across time and clades: Validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine

    PubMed Central

    Levitz, Lauren; Koita, Ousmane A.; Sangare, Kotou; Ardito, Matthew T.; Boyle, Christine M.; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M.; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D.; De Groot, Anne S.

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the “Achilles’ heel” of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. PMID:23102976

  17. Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1).

    PubMed

    Shigeta, M; Hasegawa, A; Hamada, Y; Koyama, K

    2000-07-01

    The zona pellucida (ZP) of mammalian oocytes forms an extracellular matrix composed of three major glycoproteins and plays an important role in sperm-zona interactions. As ZP had a strong organ-specific but species-cross-reactive antigenicity and passive or active immunization with ZP antigens could impair fertilization, the possibility of developing a immunocontraceptive vaccine has been extensively studied. Studies on active immunization with porcine ZP (pZP) that contain B cell epitopes and T cell epitopes demonstrated that a temporary infertility could be induced along with the elevation of antibody titers, but it was always associated with ovarian failure. This could be due to the oophoritis by activation of pathogenic T cell immunity. It is the general consideration that any adverse effects by vaccination should be avoided for an immunocontraception. From this point of view, the analysis of B cell epitopes of pZP protein would be helpful for construction of a safe immunocontraceptive vaccine with zona antigens. We determined the amino acid sequence of the B epitope in the pZP1 protein by using a monoclonal antibody (MAb-5H4) that possesses a fertilization blocking ability. In addition, antiserum raised to the epitope sequence was revealed to block in vitro fertilization of homologous animal species.

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. A comparison of two methods for T cell epitope mapping: “cell free” in vitro versus immunoinformatics

    PubMed Central

    Messitt, Timothy J.; Terry, Frances; Moise, Leonard; Martin, William

    2014-01-01

    Background Methods for identifying physiologically relevant T-cell epitopes are critically important for development of vaccines and the design of therapeutic proteins. As the number of proteins that are being evaluated for putative immunogenicity expands, rapid and accurate tools are in great demand. Several methods to identify T-cell epitopes have been developed, the most recent of which is a cell free system consisting of a minimal set of proteases incubated with HLA DRB1*0101, HLA-DM and whole antigen. Isolation and sequencing of the HLA bound peptides using mass spectrometry allows for the prospective identification of immunodominant T-cell epitopes. Results We present here, a comparison of this cell free in vitro antigen processing system to an immunoinformatics approach using the EpiMatrix algorithm. Our comparison reveals that in addition to identifying a similar set of epitopes to the cell-free system, the immunoinformatics approach prospectively identifies more HLA-DRB1*0101 epitopes and can simultaneously analyze multiple HLA alleles. Conclusions Although the cell-free system incorporates antigen processing and MHC binding, the immunoinformatics approach identifies many validated epitopes with a very high degree of accuracy and can be performed much faster with far fewer resources. PMID:25346774

  1. Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in myasthenia gravis.

    PubMed Central

    Marx, A.; Kirchner, T.; Hoppe, F.; O'Connor, R.; Schalke, B.; Tzartos, S.; Müller-Hermelink, H. K.

    1989-01-01

    Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2468286

  2. Unconventional T-cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage.

    PubMed

    Falconer, Jane; Mahida, Rahul; Venkatesh, Divya; Pearson, Jeffrey; Robinson, John H

    2016-04-01

    It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage.

  3. Harnessing Computational Biology for Exact Linear B-Cell Epitope Prediction: A Novel Amino Acid Composition-Based Feature Descriptor.

    PubMed

    Saravanan, Vijayakumar; Gautham, Namasivayam

    2015-10-01

    Proteins embody epitopes that serve as their antigenic determinants. Epitopes occupy a central place in integrative biology, not to mention as targets for novel vaccine, pharmaceutical, and systems diagnostics development. The presence of T-cell and B-cell epitopes has been extensively studied due to their potential in synthetic vaccine design. However, reliable prediction of linear B-cell epitope remains a formidable challenge. Earlier studies have reported discrepancy in amino acid composition between the epitopes and non-epitopes. Hence, this study proposed and developed a novel amino acid composition-based feature descriptor, Dipeptide Deviation from Expected Mean (DDE), to distinguish the linear B-cell epitopes from non-epitopes effectively. In this study, for the first time, only exact linear B-cell epitopes and non-epitopes have been utilized for developing the prediction method, unlike the use of epitope-containing regions in earlier reports. To evaluate the performance of the DDE feature vector, models have been developed with two widely used machine-learning techniques Support Vector Machine and AdaBoost-Random Forest. Five-fold cross-validation performance of the proposed method with error-free dataset and dataset from other studies achieved an overall accuracy between nearly 61% and 73%, with balance between sensitivity and specificity metrics. Performance of the DDE feature vector was better (with accuracy difference of about 2% to 12%), in comparison to other amino acid-derived features on different datasets. This study reflects the efficiency of the DDE feature vector in enhancing the linear B-cell epitope prediction performance, compared to other feature representations. The proposed method is made as a stand-alone tool available freely for researchers, particularly for those interested in vaccine design and novel molecular target development for systems therapeutics and diagnostics: https://github.com/brsaran/LBEEP.

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

    PubMed

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

    2014-05-30

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2006-05-26

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

  7. Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus.

    PubMed

    Gautam, Samir; Kim, Taehan; Lester, Evan; Deep, Deeksha; Spiegel, David A

    2016-01-15

    Staphylococcus aureus is a Gram-positive bacterial pathogen that produces a range of infections including cellulitis, pneumonia, and septicemia. The principle mechanism in antistaphylococcal host defense is opsonization with antibodies and complement proteins, followed by phagocytic clearance. Here we use a previously developed technique for installing chemical epitopes in the peptidoglycan cell wall to show that surface glycopolymers known as wall teichoic acids conceal cell wall epitopes, preventing their recognition and opsonization by antibodies. Thus, our results reveal a previously unrecognized immunoevasive role for wall teichoic acids in S. aureus: repulsion of peptidoglycan-targeted antibodies.

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

    PubMed

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

    2015-11-11

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

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

    PubMed

    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

    2017-10-02

    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, CX3CR1, 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.

  10. Live Cell Characterization of DNA Aggregation Delivered through Lipofection

    PubMed Central

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

    2015-01-01

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation. PMID:26013547

  11. Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

    PubMed

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A; Gratton, Enrico; Jones, Mark R

    2015-05-27

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

  12. Vertical T cell immunodominance and epitope entropy determine HIV-1 escape

    PubMed Central

    Liu, Michael K.P.; Hawkins, Natalie; Ritchie, Adam J.; Ganusov, Vitaly V.; Whale, Victoria; Brackenridge, Simon; Li, Hui; Pavlicek, Jeffrey W.; Cai, Fangping; Rose-Abrahams, Melissa; Treurnicht, Florette; Hraber, Peter; Riou, Catherine; Gray, Clive; Ferrari, Guido; Tanner, Rachel; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; B, CHAVI Core; Cohen, Myron; Karim, Salim S. Abdool; Haynes, Barton; Borrow, Persephone; Perelson, Alan S.; Shaw, George M.; Hahn, Beatrice H.; Williamson, Carolyn; Korber, Bette T.; Gao, Feng; Self, Steve; McMichael, Andrew; Goonetilleke, Nilu

    2012-01-01

    HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance. PMID:23221345

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

    PubMed Central

    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 field has evolved significantly. Methods have now been developed that produce highly accurate binding predictions for many alleles and integrate both proteasomal cleavage and transport events. Moreover have so-called pan-specific methods been developed, which allow for prediction of peptide binding to MHC alleles characterized by limited or no peptide binding data. Most of the developed methods are publicly available, and have proven to be very useful as a shortcut in epitope discovery. Here, we will go through some of the history of sequence-based predictions of helper as well as cytotoxic T cell epitopes. We will focus on some of the most accurate methods and their basic background. PMID:21067545

  14. Mapping of SLE-specific Sm B cell epitopes using murine monoclonal antibodies.

    PubMed

    Pruijn, G J; Schoute, F; Thijssen, J P; Smeenk, R J; van Venrooij, W J

    1997-04-01

    In this study we have used a number of monoclonal antibodies with various anti-Sm specificities originating from MRL/lpr mice to map B cell epitopes of the Sm-B/B' and Sm-D1 proteins. Selection of Sm-B subfragments reactive with the Sm-B/B'-specific monoclonal antibody ANA125 from a DNaseI fragment expression library revealed that the epitope recognized by this monoclonal antibody is located between amino acids 146 and 158: GRGTVAAAAAAAT. The epitopes recognized by two distinct Sm-D1-specific monoclonal antibodies, 7.13 and ANA127, appeared to be located in the carboxy-terminal region of the protein as revealed by immunoprecipitation of in vitro translated deletion mutants of Sm-D1. These epitopes are probably identical and not simply composed of a GR repeat, which is a characteristic feature of this part of the protein. Immunoprecipitation of in vitro translated deletion mutants of both Sm-B and Sm-D1 was also employed to determine the sequence requirements for recognition by two monoclonal antibodies that are cross-reactive with several Sm proteins, Y12 and ANA128. The epitope recognized by these two monoclonal antibodies is probably also identical and composed by the juxtaposition of several regions in the folded protein. The low, but significant, level of immunoprecipitation of truncated versions of both Sm-B and Sm-D1, suggests that the Sm domain, which is shared by all Sm proteins, in particular the amino-terminal part of the Sm1 motif of Sm-B and Sm-D1, plays an important role in formation of the cross-reactive epitope and might contribute to cross-reactivity with other Sm proteins. The results of immunoprecipitation experiments with cellular extracts show that the epitopes recognized by all anti-Sm monoclonal antibodies used in this study are accessible in the assembled snRNPs.

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

    PubMed

    Zheng, Wei; Ruan, Jishou; Hu, Gang; Wang, Kui; Hanlon, Michelle; Gao, Jianzhao

    2015-01-01

    The prediction of conformational b-cell epitopes plays an important role in immunoinformatics. Several computational methods are proposed on the basis of discrimination determined by the solvent-accessible surface between epitopes and non-epitopes, but the performance of existing methods is far from satisfying. In this paper, depth functions and the k-th surface convex hull are used to analyze epitopes and exposed non-epitopes. On each layer of the protein, we compute relative solvent accessibility and four different types of depth functions, i.e., Chakravarty depth, DPX, half-sphere exposure and half space depth, to analyze the location of epitopes on different layers of the proteins. We found that conformational b-cell epitopes are rich in charged residues Asp, Glu, Lys, Arg, His; aliphatic residues Gly, Pro; non-charged residues Asn, Gln; and aromatic residue Tyr. Conformational b-cell epitopes are rich in coils. Conservation of epitopes is not significantly lower than that of exposed non-epitopes. The average depths (obtained by four methods) for epitopes are significantly lower than that of non-epitopes on the surface using the Wilcoxon rank sum test. Epitopes are more likely to be located in the outer layer of the convex hull of a protein. On the benchmark dataset, the cumulate 10th convex hull covers 84.6% of exposed residues on the protein surface area, and nearly 95% of epitope sites. These findings may be helpful in building a predictor for epitopes.

  16. Human CD4+ T Cell Epitopes from Vaccinia Virus Induced by Vaccination or Infection

    PubMed Central

    Calvo-Calle, J. Mauricio; Strug, Iwona; Nastke, Maria-Dorothea; Baker, Stephen P; Stern, Lawrence J

    2007-01-01

    Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4+ T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4+ T cell responses have been poorly characterized, and CD4+ T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens. PMID:17937498

  17. Titrating T-cell epitopes within self-assembled vaccines optimizes CD4+ helper T cell and antibody outputs.

    PubMed

    Pompano, Rebecca R; Chen, Jianjun; Verbus, Emily A; Han, Huifang; Fridman, Arthur; McNeely, Tessie; Collier, Joel H; Chong, Anita S

    2014-11-01

    Epitope content plays a critical role in determining T-cell and antibody responses to vaccines, biomaterials, and protein therapeutics, but its effects are nonlinear and difficult to isolate. Here, molecular self-assembly is used to build a vaccine with precise control over epitope content, in order to finely tune the magnitude and phenotype of T helper and antibody responses. Self-adjuvanting peptide nanofibers are formed by co-assembling a high-affinity universal CD4+ T-cell epitope (PADRE) and a B-cell epitope from Staphylococcus aureus at specifiable concentrations. Increasing the PADRE concentration from micromolar to millimolar elicited bell-shaped dose-responses that are unique to different T-cell populations. Notably, the epitope ratios that maximize T follicular helper and antibody responses differed by an order of magnitude from those that maximized Th1 or Th2 responses. Thus, modular materials assembly provides a means of controlling epitope content and efficiently skewing the adaptive immune response in the absence of exogenous adjuvant; this approach may contribute to the development of improved vaccines and immunotherapies.

  18. Titrating T cell Epitopes within Self-Assembled Vaccines Optimizes CD4+ Helper T Cell and Antibody Outputs

    PubMed Central

    Chen, Jianjun; Verbus, Emily A.; Han, Huifang; Fridman, Arthur; McNeely, Tessie; Collier, Joel H.; Chong, Anita S.

    2014-01-01

    Epitope content plays a critical role in determining T cell and antibody responses to vaccines, biomaterials, and protein therapeutics, but its effects are nonlinear and difficult to isolate. Here, molecular self-assembly was used to build a vaccine with precise control over epitope content, in order to finely tune the magnitude and phenotype of T helper and antibody responses. Self-adjuvanting peptide nanofibers were formed by co-assembling a high-affinity universal CD4+ T cell epitope (PADRE) and a B cell epitope from Staphylococcus aureus at specifiable concentrations. Increasing the PADRE concentration from μM to mM elicited bell-shaped dose-responses that were unique to different T cell populations. Notably, the epitope ratios that maximized T follicular helper and antibody responses differed by an order of magnitude from those that maximized Th1 or Th2 responses. Thus, modular materials assembly provides a means of controlling epitope content and efficiently skewing the adaptive immune response in the absence of exogenous adjuvant; this approach may contribute to the development of improved vaccines and immunotherapies. PMID:24923735

  19. Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection ▿

    PubMed Central

    Sacha, Jonah B.; Buechler, Matthew B.; Newman, Laura P.; Reed, Jason; Wallace, Lyle T.; Loffredo, John T.; Wilson, Nancy A.; Watkins, David I.

    2010-01-01

    The kinetics of CD8+ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8+ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4+ T cells early after SIV infection. PMID:20686015

  20. Multiple B- and T-cell epitopes on a major allergen of Kentucky Bluegrass pollen.

    PubMed Central

    Zhang, L; Yang, M; Chong, P; Mohapatra, S S

    1996-01-01

    The B- and T-cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme-linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin-, and nine to 13 human IgG- and five to seven human IgE-reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen-specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross-recognition of different peptides at the C-terminus of rKBG60 by antibodies to neighbouring or non-overlapping peptides suggest that the C-terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T-cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B- and T-cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C-terminus of this antigen. PMID:8698392

  1. Orchestration of CD4 T cell epitope preferences after multi-peptide immunization

    PubMed Central

    Tung, Jacqueline; Sant, Andrea J.

    2013-01-01

    A detailed understanding of the molecular and cellular mechanisms that underlie epitope preferences in T cell priming is important for vaccines designed to elicit a broad T cell response. Protein vaccinations generally elicit CD4 T cell responses that are skewed toward a small fraction of epitopes, a phenomenon known as immunodominance. This characteristic of T cell responses, that limits the diversity of CD4 T cell recognition, is generally attributed to intracellular antigen processing. However, we recently discovered that immunodominance hierarchies persist even after vaccination with synthetic peptides. In this study, we probed the regulatory mechanisms that cause diminished CD4 T cell responses to subdominant peptides after such multi-peptide immunization in mice. We have found that the delivery of subdominant and dominant epitopes on separate dendritic cells rescues expansion of less favored CD4 T cells. Furthermore, through the use of genetic models and inhibitors, we have found that selective losses in CD4 T cell responses are mediated by an IFN-γ-induced pathway, involving indoleamine 2,3-dioxygenase (IDO), and that regulatory T cell (Treg) activities may also regulate preferences in CD4 T cell specificity. We propose that after multi-peptide immunization, the expansion and differentiation of dominant T cells initiate complex regulatory events that determine the final peptide specificity of the elicited CD4 T cell response. PMID:23772029

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

    PubMed

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

    2006-06-15

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

  3. Delivering factors for reprogramming a somatic cell to pluripotency.

    PubMed

    Um, Soong Ho

    2012-05-01

    An adult cell originates from stem cell. The stem cell is usually categorized into three species including an embryonic stem cell (ESc), an adult stem cell, and an induced stem cell (iPSc). iPSc features pluripotency, which is meant to be differentiated into any types of cells. Accordingly, it is much attractive to anyone who pursuit a regenerative medicine, owing to the potential almighty. They are simply produced by reprogramming a somatic cell via a transfer of transcription factors. The efficiency and productivity of iPS are considerably subject to delivering methods of exogenous genes into a variety of targeted mammalians. Conventional and well-run gene delivery techniques have been reviewed here. This details the methods and principles of delivery factors and provides an overview of the research, with an emphasis on their potential for use as clinical therapeutic platforms.

  4. CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES, CHLOROPHYTA)(1).

    PubMed

    Estevez, José M; Leonardi, Patricia I; Alberghina, Josefina S

    2008-10-01

    Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)-β-glucans in the form of crystalline cellulose together with a variable degree of Me-esterified homogalacturonans (HGs) and hydroxyproline-rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan-protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM-5, LM-6, and CCRC-M2) was found, suggesting the presence of rhamnogalacturonan I (RG-I)-like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O-glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few-celled to complex multicelled organisms.

  5. Selective Differentiation of Neural Progenitor Cells by High-Epitope Density Nanofibers

    NASA Astrophysics Data System (ADS)

    Silva, Gabriel A.; Czeisler, Catherine; Niece, Krista L.; Beniash, Elia; Harrington, Daniel A.; Kessler, John A.; Stupp, Samuel I.

    2004-02-01

    Neural progenitor cells were encapsulated in vitro within a three-dimensional network of nanofibers formed by self-assembly of peptide amphiphile molecules. The self-assembly is triggered by mixing cell suspensions in media with dilute aqueous solutions of the molecules, and cells survive the growth of the nanofibers around them. These nanofibers were designed to present to cells the neurite-promoting laminin epitope IKVAV at nearly van der Waals density. Relative to laminin or soluble peptide, the artificial nanofiber scaffold induced very rapid differentiation of cells into neurons, while discouraging the development of astrocytes. This rapid selective differentiation is linked to the amplification of bioactive epitope presentation to cells by the nanofibers.

  6. Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

    PubMed

    Kalaiselvan, Sagadevan; Sankar, Sathish; Ramamurthy, Mageshbabu; Ghosh, Asit Ranjan; Nandagopal, Balaji; Sridharan, Gopalan

    2017-01-20

    Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 9999: 1-5, 2017. © 2017 Wiley Periodicals, Inc.

  7. In silico prediction of B cell epitopes of the extracellular domain of insulin-like growth factor-1 receptor

    PubMed Central

    Bayrami, Vahid; Keyhanfar, Mehrnaz; Mohabatkar, Hassan; Mahdavi, Manijeh; Moreau, Violaine

    2016-01-01

    The insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane receptor with tyrosine kinase activity. The receptor plays a critical role in cancer. Using monoclonal antibodies (MAbs) against the IGF-1R, typically blocks ligand binding and enhances down-regulation of the cell-surface IGF-1R. Some MAbs such as cixutumumab are under clinical trial investigation. Targeting multiple distinct epitopes on IGF-1R, might be an effective strategy to inhibit IGF-1R pathway in cancer. In this study, new linear B cell epitopes for the extracellular domains of IGF-1R were predicted by in silico methods using a combination of linear B cell epitope prediction web servers such as ABCpred, Bepired, BCPREDs, Bcepred and Elliprro. Moreover, Discotope, B- pred and PEPOP web server tools were employed to predict new conformational B cell epitopes. In contrast to previously reported epitopes from extracellular region of the IGF-1R, we predicted new linear P8: (RQPQDGYLYRHNYCSK) and conformational Pc4: (HYYYAGVCVPACPPNTYRFE), Ppc6: (KMCPSTGKRENNESAPDNDT) and Ppc20: (ANILSAESSDSEFMQEPSGFI) epitopes. These epitopes are useful for further study as peptide antigens to actively immune host animals to develop new MAbs. Furthermore, the epitopes can be used in peptide-based cancer vaccines design. PMID:28261624

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

    PubMed Central

    2010-01-01

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

  9. Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE

    PubMed Central

    Shetty, Aparna; Gupta, Sheena G.; Varrin-Doyer, Michel; Weber, Martin S.; Prod'homme, Thomas; Molnarfi, Nicolas; Ji, Niannian; Nelson, Patricia A.; Patarroyo, Juan C.; Schulze-Topphoff, Ulf; Fogal, Stephen E.; Forsthuber, Thomas; Sobel, Raymond A.; Bernard, Claude C.A.; Slavin, Anthony J.

    2014-01-01

    Objective: Studies evaluating T-cell recognition of myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), have focused mostly on its 117 amino acid (aa) extracellular domain, especially peptide (p) 35-55. We characterized T-cell responses to the entire 218 aa MOG sequence, including its transmembrane and cytoplasmic domains. Methods: T-cell recognition in mice was examined using overlapping peptides and intact full-length mouse MOG. EAE was evaluated by peptide immunization and by adoptive transfer of MOG epitope-specific T cells. Frequency of epitope-specific T cells was examined by ELISPOT. Results: Three T-cell determinants of MOG were discovered in its transmembrane and cytoplasmic domains, p119–132, p181–195, and p186–200. Transmembrane MOG p119-132 induced clinical EAE, CNS inflammation, and demyelination as potently as p35-55 in C57BL/6 mice and other H-2b strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2b strains, including Biozzi, NOD, and PL/J. MOG p119-132–specific T cells produced Th1 and Th17 cytokines and transferred EAE to wild-type recipient mice. After immunization with full-length MOG, a significantly higher frequency of MOG-reactive T cells responded to p119-132 than to p35-55, demonstrating that p119-132 is an immunodominant encephalitogenic epitope. MOG p181-195 did not cause EAE, and MOG p181-195–specific T cells could not transfer EAE into wild-type or highly susceptible T- and B-cell–deficient mice. Conclusions: Transmembrane and cytoplasmic domains of MOG contain immunodominant T-cell epitopes in EAE. A CNS autoantigen can also contain nonpathogenic stimulatory T-cell epitopes. Recognition that a myelin antigen contains multiple encephalitogenic and nonencephalitogenic determinants may have implications for therapeutic development in MS. PMID:25340074

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

    PubMed Central

    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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    1996-01-01

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

  13. T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.

    PubMed

    Widhe, M; Ekerfelt, C; Jarefors, S; Skogman, B H; Peterson, E M; Bergström, S; Forsberg, P; Ernerudh, J

    2009-08-01

    We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

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

    PubMed

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

    2012-11-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

    The interaction between antibodies and antigens is one of the most important immune system mechanisms for clearing infectious organisms from the host. Antibodies bind to antigens at sites referred to as B-cell epitopes. Identification of the exact location of B-cell epitopes is essential in several 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 of B-cell epitopes has been moderate. Several issues regarding the evaluation data sets may however have led to the performance values being underestimated: Rarely, all potential epitopes have been mapped on an antigen, and antibodies are generally raised against the antigen in a given biological context not against the antigen monomer. Improper dealing with these aspects leads to many artificial false positive predictions and hence to incorrect low performance values. To demonstrate the impact of proper benchmark definitions, we here present an updated version of the DiscoTope method incorporating a novel spatial neighborhood definition and half-sphere exposure as surface measure. Compared to other state-of-the-art prediction methods, Discotope-2.0 displayed improved performance both in cross-validation and in independent evaluations. Using DiscoTope-2.0, we assessed the impact on performance when using proper benchmark definitions. For 13 proteins in the training data set where sufficient biological information was available to make a proper benchmark redefinition, the average AUC performance was improved from 0.791 to 0.824. Similarly, the average AUC performance on an independent 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

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

    NASA Astrophysics Data System (ADS)

    Logean, Antoine; Rognan, Didier

    2002-04-01

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

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

    PubMed

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

    2015-01-30

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

  19. Superimposed epitopes restricted by the same HLA molecule drive distinct HIV-specific CD8+ T cell repertoires.

    PubMed

    Sun, Xiaoming; Fujiwara, Mamoru; Shi, Yi; Kuse, Nozomi; Gatanaga, Hiroyuki; Appay, Victor; Gao, George F; Oka, Shinichi; Takiguchi, Masafumi

    2014-07-01

    Superimposed epitopes, in which a shorter epitope is embedded within a longer one, can be presented by the same HLA class I molecule. CD8(+) CTL responses against such epitopes and the contribution of this phenomenon to immune control are poorly characterized. In this study, we examined HLA-A*24:02-restricted CTLs specific for the superimposed HIV Nef epitopes RYPLTFGWCF (RF10) and RYPLTFGW (RW8). Unexpectedly, RF10-specific and RW8-specific CTLs from HIV-1-infected HLA-A*24:02+ individuals had no overlapping Ag reactivity or clonotypic compositions. Single-cell TCR sequence analyses demonstrated that RF10-specific T cells had a more diverse TCR repertoire than did RW8-specific T cells. Furthermore, RF10-specific CTLs presented a higher Ag sensitivity and HIV suppressive capacity compared with RW8-specific CTLs. Crystallographic analyses revealed important structural differences between RF10- and RW8-HLA-A*24:02 complexes as well, with featured and featureless conformations, respectively, providing an explanation for the induction of distinct T cell responses against these epitopes. The present study shows that a single viral sequence containing superimposed epitopes restricted by the same HLA molecule could elicit distinct CD8+ T cell responses, therefore enhancing the control of HIV replication. This study also showed that a featured epitope (e.g., RF10) could drive the induction of T cells with high TCR diversity and affinity. Copyright © 2014 by The American Association of Immunologists, Inc.

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

    PubMed

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

    1999-08-15

    Although atopic allergy affects 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.

  1. Parasite stage-specific recognition of endogenous Toxoplasma gondii derived CD8+ T cell epitopes

    PubMed Central

    Frickel, Eva-Maria; Sahoo, Nivedita; Hopp, Johnathan; Gubbels, Marc-Jan; Craver, Mary Patricia J.; Knoll, Laura J.; Ploegh, Hidde L.; Grotenbreg, Gijsbert M.

    2016-01-01

    Background BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. IFN-γ producing CD8+ T cells provide essential protection against T. gondii, but the epitopes recognized have so far remained elusive. Methods We employed caged MHC molecules to generate ~ 250 H-2Ld tetramers and distinguish T. gondii-specific CD8+ T cells in BALB/c mice. Results We identify two T. gondii specific H-2Ld-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2Ld/GRA4 reactive T cells from multiple organ sources predominate 2 weeks after infection, while the reactivity of the H-2Ld/ROP7 T cells peaks 6–8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection show altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Conclusions Our results shed light on the identity and the parasite stage-specificity of two CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii. PMID:18922097

  2. Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8+ T cell epitopes.

    PubMed

    Frickel, Eva-Maria; Sahoo, Nivedita; Hopp, Johnathan; Gubbels, Marc-Jan; Craver, Mary Patricia J; Knoll, Laura J; Ploegh, Hidde L; Grotenbreg, Gijsbert M

    2008-12-01

    BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8+ T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. We employed caged major histocompatibility complex molecules to generate approximately 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8+ T cells in BALB/c mice. We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Our results shed light on the identity and the parasite stage-specificity of 2 CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.

  3. Functional Properties and Epitope Characteristics of T-Cells Recognizing Natural HIV-1 Variants

    PubMed Central

    Malhotra, U; Nolin, J.; Horton, H; Li, F; Corey, L; Mullins, JI; McElrath, MJ

    2009-01-01

    To understand how broad recognition of HIV-1 variants may be achieved we examined T-cell reactivity in newly-infected persons as well as vaccine recipients to a broad spectrum of potential T-cell epitope (PTE) variants containing conservative, semi-conservative and non-conservative amino acid substitutions. Among early-infected persons T-cells recognized epitope variants with one substitution at a significantly higher frequency versus those with two (P=0.0098) and three (P=0.0125) substitutions. Furthermore T cells recognized variants containing conservative substitutions at a higher frequency versus those containing semi-conservative (P=0.0029) and non-conservative (P<0.0001) substitutions. Similar effects were observed on recognition of variants by vaccine induced T-cells. Moreover even when variants were recognized, the IFN-γ and granzyme B responses as well as T-cell proliferation were of lower magnitude. Finally, we show that epitope distribution is strongly influenced by both processing preferences and amino acid entropy. We conclude that induction of broad immunity is likely to require immunogen sequences that encompass multiple variants. However, cost-effective design of peptide and sequence based vaccine immunogens that provide maximal coverage of circulating sequences may be achieved through emphasis on virus domains likely to be T-cell targets. PMID:19747576

  4. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Veillette, Maxime; Désormeaux, Anik; Medjahed, Halima; Gharsallah, Nour-Elhouda; Coutu, Mathieu; Baalwa, Joshua; Guan, Yongjun; Lewis, George; Ferrari, Guido; Hahn, Beatrice H; Haynes, Barton F; Robinson, James E; Kaufmann, Daniel E; Bonsignori, Mattia; Sodroski, Joseph; Finzi, Andrés

    2014-03-01

    Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

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

    PubMed

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

    2009-06-12

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

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

    PubMed Central

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

    2009-01-01

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

  7. Identification of Cross-Reactive Norovirus CD4+ T Cell Epitopes

    PubMed Central

    LoBue, Anna D.; Lindesmith, Lisa C.; Baric, Ralph S.

    2010-01-01

    Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4+ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans. PMID:20573810

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

    PubMed Central

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

    2002-01-01

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

  9. Recognition of a common mycobacterial T-cell epitope in MPB59 of Mycobacterium bovis.

    PubMed Central

    Lightbody, K A; Girvin, R M; Pollock, D A; Mackie, D P; Neill, S D; Pollock, J M

    1998-01-01

    Bovine tuberculosis, which persists as a residual level of infection in many European countries, has implications not only for the economy of farming communities but also for human health. The aim of this study was to identify a common mycobacterial antigen which was recognized in bovine tuberculosis and to characterize the response to this antigen at the epitope level. A T-cell clone, phenotype CD4+, raised from an animal experimentally infected with Mycobacterium bovis was shown to proliferate in response to a panel of sonicates derived from different mycobacterial species indicating recognition of an antigen with broad specificity. This antigen was subsequently shown to be MPB59. Recognition of MPB59 at the epitope level was determined in experimental and field cases of bovine tuberculosis using a panel of synthetic peptides (20-mers with 10-residue overlaps) incorporating the signal sequence and mature protein. The results showed that in vitro interferon-gamma was predominantly produced in response to adjacent peptides numbers 10 and 11, suggesting that the dominant epitope was contained in the overlap, correlating to residues 101-110 (YYQSGLSIVM). This epitope was recognized by 54% of tuberculous cattle of mixed breeds, which suggests that it may be genetically permissive in terms of major histocompatibility complex presentation. Sequence analysis confirmed that there were only minor differences in the amino acid composition within this region for various mycobacterial species, which could explain the common T-cell recognition described in this study. Common recognition of this epitope indicates that it would have limited potential for use as a diagnostic reagent per se but may have potential for inclusion in a subunit vaccine. PMID:9640240

  10. Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  12. Vaccination with Altered Peptide Ligands of a Plasmodium berghei Circumsporozoite Protein CD8 T-Cell Epitope: A Model to Generate T Cells Resistant to Immune Interference by Polymorphic Epitopes

    PubMed Central

    Minigo, Gabriela; Flanagan, Katie L.; Slattery, Robyn M.; Plebanski, Magdalena

    2017-01-01

    Many pathogens, including the malaria parasite Plasmodium falciparum, display high levels of polymorphism within T-cell epitope regions of proteins associated with protective immunity. The T-cell epitope variants are often non-cross-reactive. Herein, we show in a murine model, which modifies a protective CD8 T-cell epitope from the circumsporozoite protein (CS) of Plasmodium berghei (SYIPSAEKI), that simultaneous or sequential co-stimulation with two of its putative similarly non-cross-reactive altered peptide ligand (APL) epitopes (SYIPSAEDI or SYIPSAEAI) has radically different effects on immunity. Hence, co-immunization or sequential stimulation in vivo of SYIPSAEKI with its APL antagonist SYIPSAEDI decreases immunity to both epitopes. By contrast, co-immunization with SYIPSAEAI has no apparent initial effect, but it renders the immune response to SYIPSAEKI resistant to being turned off by subsequent immunization with SYIPSAEDI. These results suggest a novel strategy for vaccines that target polymorphic epitopes potentially capable of mutual immune interference in the field, by initiating an immune response by co-immunization with the desired index epitope, together with a carefully selected “potentiator” APL peptide. PMID:28261200

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

    PubMed

    Shorter, Shayla K; Schnell, Frederick J; McMaster, Sean R; Pinelli, David F; Andargachew, Rakieb; Evavold, Brian D

    2016-01-01

    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.

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

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    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. PMID:26915099

  15. [Identification and evaluation of T cell epitopes of Rv0585c from Mycobacterium tuberculosis].

    PubMed

    Wang, X X; Chen, X; Li, Y Q; Xiao, T Y; Jiang, Y; Li, M C; Liu, H C; Wan, K L

    2017-05-10

    Objective: To investigate the human T cell epitopes of Mycobacterium (M.) tuberculosis Rv0585c protein antigen and their immunogenicity and provide evidence for the development of specific tuberculosis immune diagnostic techniques and tuberculosis vaccine. Methods: We synthesized peptides from M. tuberculosis Rv0585c protein antigen predicted by TE-predict and IEDB human T cell epitope prediction tool. The cellular immunoreactivity of the predicted peptides was evaluated through ELISpot assay with the peripheral blood monouclear cells (PBMC) of clinical tuberculosis patients. In animal experiments, BALB/c mice were respectively immunized with high dose (100 μg/mice) and low dose (50 μg/mice) of the peptides of Rv0585c, at the same time, high dose (50 μg/mice) and low dose (20 μg/mice) of Ag85B protein were used in positive control group. The levels of IFN-γ, IL-2, IL-4 and IL-10 were tested with ELISA kit respectively. Results: By means of bioinformatics technique, 66 human T cell epitopes of Rv0585c were predicted, from which9 peptides concentrated epitopes were synthesized for the animal immune experiments. Peptides P10110, P10112 and P10117 were confirmed to be antigenic. The sensitivity and specificity of P10110, P10112 and P10117 were 14.00%, 12.00%, 6.00% and 100.00%, 100.00%, 97.96% respectively when they were used as diagnostic reagents of tuberculosis. The sensitivity and specificity were 22.00% and 97.96% when the epitopes were combined together. The results of animal immunity test showed that high levels of cytokines IFN-γ, IL-2, IL-4 and IL-10 were induced by high and low dose of P10110, and high levels of IFN-γ、IL-2 and IL-10 were induced by high and low dose of P10112, which were much higher than that in negative controls, respectively (P<0.001). Conclusion: Rv0585c, including its human T cell epitopes, has good immunogenicity and immunoreactivity, stimulating the body to produce a stronger cellular immune response and has better potential

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  18. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    NASA Astrophysics Data System (ADS)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  19. Estimation and extraction of B-cell linear epitopes predicted by mathematical morphology approaches.

    PubMed

    Chang, Hao-Teng; Liu, Chih-Hong; Pai, Tun-Wen

    2008-01-01

    B-cell epitope prediction facilitates the design and synthesis of short peptides for various immunological applications. Several algorithms have been developed to predict B-cell linear epitopes (LEs) from primary sequences of antigens, providing important information for immunobiological experiments and antibody design. This paper describes two robust methods, LE prediction with/without local peak extraction (LEP-LP and LEP-NLP), based on antigenicity scale and mathematical morphology for the prediction of B-cell LEs. Previous studies revealed that LEs could occur in regions with low-to-moderate but not globally high antigenicity scales. Hence, we developed a method adopting mathematical morphology to extract local peaks from a linear combination of the propensity scales of physico-chemical characteristics at each antigen residue. Comparison among LEP-LP/LEP-NLP, BepiPred and BEPITOPE revealed that our algorithms performed better in retrieving epitopes with low-to-moderate antigenicity and achieved comparable performance according to receiver operation characteristics (ROC) curve analysis. Of the identified LEs, over 30% were unable to be predicted by BepiPred and BEPITOPE employing an average threshold of antigenicity index or default settings. Our LEP-LP method provides a bioinformatics approach for predicting B-cell LEs with low- to-moderate antigenicity. The web-based server was established at http://biotools.cs.ntou.edu.tw/lepd_antigenicity. php for free use.

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

    PubMed Central

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

    2005-01-01

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

  1. Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization.

    PubMed

    Marti, Thomas; Molberg, Oyvind; Li, Qing; Gray, Gary M; Khosla, Chaitan; Sollid, Ludvig M

    2005-01-01

    Celiac Sprue is a widely prevalent immune disease of the small intestine induced by dietary gluten intake in genetically susceptible individuals. It has been suggested that prolyl endopeptidases (PEPs) may be useful catalysts for gluten detoxification. We have investigated this hypothesis using food-grade gluten as the target antigen, and a combination of mass spectrometry and patient-derived T cells as quantitative assay systems. Spectrometric characterization of physiologically proteolyzed gluten revealed a number of 10 to 50 residue peptides containing known T cell epitopes involved in Celiac Sprue pathogenesis. Several of these peptides were multivalent, suggesting they may be potent triggers of the inflammatory response to gluten in celiac patients. Treatment of proteolyzed gluten with recombinant bacterial PEP decreased the number of potentially immunostimulatory peptides. Substantially reduced immunogenicity was also quantified in 12 of 14 intestinal polyclonal T cell lines from celiac patients. Kinetic investigations using eight T cell clones showed rapid destruction of alpha-gliadin epitopes, but less complete processing of gamma-gliadin epitopes. Given the difficulty associated with a strict lifelong gluten-exclusion diet, the ability of a single enzyme to greatly reduce the antigenic burden of grocery store gluten reinforces the case for developing oral peptidase therapy against Celiac Sprue.

  2. Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.

    PubMed

    Prickett, S R; Voskamp, A L; Phan, T; Dacumos-Hill, A; Mannering, S I; Rolland, J M; O'Hehir, R E

    2013-06-01

    Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (≤ 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays. Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations. © 2013 John Wiley & Sons Ltd.

  3. Identification of human papillomavirus-16 E6 variation in cervical cancer and their impact on T and B cell epitopes.

    PubMed

    Kumar, Anoop; Hussain, Showket; Yadav, Inderjit Singh; Gissmann, Lutz; Natarajan, K; Das, Bhudev C; Bharadwaj, Mausumi

    2015-06-15

    The infection with high-risk human papillomavirus (HR-HPV) is the most important risk factor for development of cervical cancer. The intra-type variations of HPV have different biological and pathological consequences with respect to disease progression. In the present study, six major Indian variants were experimentally identified in E6 gene of HPV-16 and showed their impact on immunogenicity by in silico methods. Four different phylogenetic lineages were observed in sequences including European (E) prototype, European variant, Asian and American Asian variant classes and complete absence of African phylogenetic lineages. On the prediction of B- and T-cell epitopes, 18 and 23 potent epitopes for MHC-II alleles, 10 potent MHC-I and 15 B-cell epitopes in each reference and variant sequence were identified. Interestingly, the presence of variation H78Y and L83V result in creation of four new epitopes for the HLA-DQA1*0101/DQB1*0501. Out of 15 B-cell predicted epitopes, three most potent epitopes were identified in both reference and variant sequence. Notably the amino acid stretch from amino acid 16-60 and 76-94 are very important for the immunological properties of E6 protein because these regions contain majority of the predicted epitopes. In future, this could control the cervical cancer by targeting these amino acid stretches for the development of HPV-16 vaccine.

  4. In silico methods for predicting T-cell epitopes: Dr Jekyll or Mr Hyde?

    PubMed

    Gowthaman, Uthaman; Agrewala, Javed N

    2009-10-01

    In silico tools offer an attractive alternative strategy to the cumbersome experimental approaches to identify T-cell epitopes. These computational tools have metamorphosed over the years into complex algorithms that attempt to efficiently predict the binding of a plethora of peptides to HLA alleles. In recent years, the scientific community has embraced these techniques to reduce the burden of wet-laboratory experimentation. Although there are some splendid examples of the utility of these methods, there are also evidences where they fall short and remain inconsistent. Hence, are these computational tools 'Dr Jekyll' or 'Mr Hyde' to the researcher, who wishes to utilize them intrepidly? This article reviews the progress and pitfalls of the in silico tools that identify T-cell epitopes.

  5. Correlating low-similarity peptide sequences and HIV B-cell epitopes.

    PubMed

    Kanduc, Darja; Serpico, Rosario; Lucchese, Alberta; Shoenfeld, Yehuda

    2008-02-01

    Although a large number of human immunodeficiency virus-1 (HIV-1) derived B-cell epitopes has been experimentally identified, the structural requirements underlying HIV humoral immune response remain unknown. Here, we review the current literature on HIV B-cell epitopes as catalogued in the www.hiv.lanl.gov/content/immunology website, searching for common structural and/or functional immunogenic motifs. The analysis of HIV antibody data documents that the linear determinants recognized by human or murine humoral immune responses, are (or harbor) pentapeptide fragments with no or only very low similarity to the respective host proteome. The present literature analysis provides relevant insights that may be applied to design anti-HCV therapeutic approaches exempt from autoimmune collateral effects.

  6. Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV.

    PubMed

    Chiu, Christopher; McCausland, Megan; Sidney, John; Duh, Fuh-Mei; Rouphael, Nadine; Mehta, Aneesh; Mulligan, Mark; Carrington, Mary; Wieland, Andreas; Sullivan, Nicole L; Weinberg, Adriana; Levin, Myron J; Pulendran, Bali; Peters, Bjoern; Sette, Alessandro; Ahmed, Rafi

    2014-03-01

    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.

  7. In silico prediction of Ara h 2 T cell epitopes in peanut-allergic children.

    PubMed

    Pascal, M; Konstantinou, G N; Masilamani, M; Lieberman, J; Sampson, H A

    2013-01-01

    Despite the frequency and severity of peanut allergy, the only approved treatment is strict avoidance. Different types of immunotherapy with crude peanut extract are not universally effective and have been associated with relatively high adverse reaction rates. We sought to determine whether in silico predictive algorithms were useful in identifying candidate peptides for an Ara h 2 peptide-based vaccine using peanut-allergic patients' peripheral blood mononuclear cells (PBMCs) in vitro. A human leucocyte antigen (HLA) distribution analysis was also performed. Major histocompatibility complex (MHC)-class II-binding peptides were predicted using NetMHCIIpan-2.0 and NetMHCII-2.2 algorithms. PBMCs from 80 peanut-allergic patients were stimulated with overlapping 20-mer Ara h 2 peptides. Cell supernatant cytokine profiles were evaluated by multiplex assays. HLA-DRB1* and HLA-DQB1* typing were performed. Four regions of overlapping sequences induced PBMC proliferation and predominant Th2 cytokine production. HLA genotyping showed 30 different DRB1* allele specificities and eight DQ serological specificities. The in silico analysis revealed similar relevant regions and predicted identical or similar core 9-mer epitopes to those identified in vitro. If relevant peptides, as determined by either in vitro or in silico analysis (15 peptides and 9 core epitopes respectively), were used in a peptide-based vaccine, they would cover virtually all subjects in the cohort studied. Four dominant regions in Ara h 2 have been identified, containing sequences that could serve as potential candidates for peptide-based immunotherapy. MHC-class II-based T cell epitope prediction algorithms for HLA-DR and -DQ loci accurately predicted Ara h 2 T cell epitopes in peanut-allergic subjects, suggesting their potential utility in a peptide-based vaccine design for food allergy. © 2012 Blackwell Publishing Ltd.

  8. Identification of class I HLA T cell control epitopes for West Nile virus.

    PubMed

    Kaabinejadian, Saghar; Piazza, Paolo A; McMurtrey, Curtis P; Vernon, Stephen R; Cate, Steven J; Bardet, Wilfried; Schafer, Fredda B; Jackson, Kenneth W; Campbell, Diana M; Buchli, Rico; Rinaldo, Charles R; Hildebrand, William H

    2013-01-01

    The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.

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

    PubMed Central

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

    2010-01-01

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

  10. Future of an “Asymptomatic” T-cell Epitope-Based Therapeutic Herpes Simplex Vaccine

    PubMed Central

    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

  11. CD4+ T cells recognize diverse epitopes within GAD65: implications for repertoire development and diabetes monitoring

    PubMed Central

    Yang, Junbao; James, Eddie A.; Sanda, Srinath; Greenbaum, Carla; Kwok, William W.

    2013-01-01

    Summary Type 1 diabetes is associated with T‐cell responses to β‐cell antigens such as GAD65. Single T‐cell epitopes have been investigated for immune monitoring with some success, but multiple epitopes may be required to fully characterize responses in all subjects. We used a systematic approach to examine the diversity of the GAD65‐specific T‐cell repertoire in subjects with DRB1*04:01 haplotypes. Using class II tetramers, we observed responses to 15 GAD65 epitopes, including five novel epitopes. The majority were confirmed to be processed and presented. Upon stimulation with peptides, GAD‐specific responses were equally broad in subjects with diabetes and healthy controls in the presence or absence of CD25+ T cells, suggesting that a susceptible HLA is sufficient to generate a potentially autoreactive repertoire. Without depleting CD25+ cells, GAD113–132 and GAD265–284 responses were significantly stronger in subjects with diabetes. Although nearly every individual responded to at least one GAD65 epitope, most were seen in less than half of the subjects tested, suggesting that multiple epitopes are recommended for immune monitoring. PMID:23228173

  12. T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles

    PubMed Central

    2012-01-01

    Introduction Anti-citrullinated peptide antibodies are found in rheumatoid arthritis (RA) patients with HLA-DRβ chains encoding the shared epitope (SE) sequence. Citrullination increases self-antigen immunogenicity, through increased binding affinity to SE-containing HLA-DR molecules. To characterise T-cell autoreactivity towards citrullinated self-epitopes, we profiled responses of SE+ healthy controls and RA patients to citrullinated and unmodified epitopes of four autoantigens. Methods We compared T-cell proliferative and cytokine responses to citrullinated and native type II collagen 1,237 to 1,249, vimentin 66 to 78, aggrecan 84 to 103 and fibrinogen 79 to 91 in six SE+ healthy controls and in 21 RA patients with varying disease duration. Cytokine-producing cells were stained after incubation with peptide in the presence of Brefeldin-A. Results Although proliferative responses were low, IL-6, IL-17 and TNF were secreted by CD4+ T cells of SE+ RA patients and healthy controls, as well as IFNγ and IL-10 secreted by RA patients, in response to citrullinated peptides. Of the epitopes tested, citrullinated aggrecan was most immunogenic. Patients with early RA were more likely to produce IL-6 in response to no epitope or to citrullinated aggrecan, while patients with longstanding RA were more likely to produce IL-6 to more than one epitope. Cytokine-producing CD4+ T cells included the CD45RO+ and CD45RO- and the CD28+ and CD28- subsets in RA patients. Conclusion Proinflammatory cytokines were produced by CD4+ T cells in SE+ individuals in response to citrullinated self-epitopes, of which citrullinated aggrecan was most immunogenic. Our data suggest that the T-cell response to citrullinated self-epitopes matures and diversifies with development of RA. PMID:22594821

  13. Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope.

    PubMed Central

    Del Val, M; Schlicht, H J; Volkmer, H; Messerle, M; Reddehase, M J; Koszinowski, U H

    1991-01-01

    The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity. Images PMID:1710286

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

    PubMed

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

    2010-07-21

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

  15. Perturbed CD8(+) T cell immunity across universal influenza epitopes in the elderly.

    PubMed

    Nguyen, Thi H O; Sant, Sneha; Bird, Nicola L; Grant, Emma J; Clemens, E Bridie; Koutsakos, Marios; Valkenburg, Sophie A; Gras, Stephanie; Lappas, Martha; Jaworowski, Anthony; Crowe, Jane; Loh, Liyen; Kedzierska, Katherine

    2017-09-19

    Influenza epidemics lead to severe illness, life-threatening complications, and deaths, especially in the elderly. As CD8(+) T cells are associated with rapid recovery from influenza, we investigated the effects of aging on antigen-specific CD8(+) T cells across the universal influenza epitopes in humans. We show that aging is characterized by altered frequencies in T cell subsets, with naive T cells being partially replaced by activated effector/memory populations. Although we observed no striking differences in TCR signaling capacity, T cells in the elderly had increased expression of transcription factors Eomes and T-bet, and such changes were most apparent in CD8(+) T cells. Strikingly, the numbers of antigen-specific CD8(+) T cells across universal influenza epitopes were reduced in the elderly, although their effector/memory phenotypes remained stable. To understand whether diminished numbers of influenza-specific CD8(+) T cells in the elderly resulted from alteration in TCR clonotypes, we dissected the TCRαβ repertoire specific for the prominent HLA-A*02:01-restricted-M158-66 (A2/M158) influenza epitope. We provide the first ex vivo data on paired antigen-specific TCRαβ clonotypes in the elderly, showing that influenza-specific A2/M158(+) TCRαβ repertoires in the elderly adults varied from those in younger adults, with the main features being a reduction in the frequency of the public TRAV27-TRBV19 TCRαβ clonotype, increased proportion of private TCRαβ signatures, broader use of TRAV and TRBV gene segments, and large clonal expansion of private TCRαβ clonotypes with longer CDR3 loops. Our study supports the development of T cell-targeted influenza vaccines that would boost the T cell compartment during life and maintain the numbers and optimal TCRαβ signatures in the elderly. © Society for Leukocyte Biology.

  16. Biodegradable nanocomposite microparticles as drug delivering injectable cell scaffolds.

    PubMed

    Wen, Yanhong; Gallego, Monica Ramos; Nielsen, Lene Feldskov; Jorgensen, Lene; Everland, Hanne; Møller, Eva Horn; Nielsen, Hanne Mørck

    2011-11-30

    Injectable cell scaffolds play a dual role in tissue engineering by supporting cellular functions and delivering bioactive molecules. The present study aimed at developing biodegradable nanocomposite microparticles with sustained drug delivery properties thus potentially being suitable for autologous stem cell therapy. Semi-crystalline poly(l-lactide/dl-lactide) (PLDL70) and poly(l-lactide-co-glycolide) (PLGA85) were used to prepare nanoparticles by the double emulsion method. Uniform and spherical nanoparticles were obtained at an average size of 270-300 nm. The thrombin receptor activator peptide-6 (TRAP-6) was successfully loaded in PLDL70 and PLGA85 nanoparticles. During the 30 days' release, PLDL70 nanoparticles showed sustainable release with only 30% TRAP-6 released within the first 15 days, while almost 80% TRAP-6 was released from PLGA85 nanoparticles during the same time interval. The release mechanism was found to depend on the crystallinity and composition of the nanoparticles. Subsequently, mPEG-PLGA nanocomposite microparticles containing PLDL70 nanoparticles were produced by the ultrasonic atomization method and evaluated to successfully preserve the intrinsic particulate properties and the sustainable release profile, which was identical to that of the nanoparticles. Good cell adhesion of the human fibroblasts onto the nanocomposite microparticles was observed, indicating the desired cell biocompatibility. The presented results thus demonstrate the development of nanocomposite microparticles tailored for sustainable drug release for application as injectable cell scaffolds.

  17. TepiTool: A Pipeline for Computational Prediction of T Cell Epitope Candidates.

    PubMed

    Paul, Sinu; Sidney, John; Sette, Alessandro; Peters, Bjoern

    2016-08-01

    Computational prediction of T cell epitope candidates is currently being used in several applications including vaccine discovery studies, development of diagnostics, and removal of unwanted immune responses against protein therapeutics. There have been continuous improvements in the performance of MHC binding prediction tools, but their general adoption by immunologists has been slow due to the lack of user-friendly interfaces and guidelines. Current tools only provide minimal advice on what alleles to include, what lengths to consider, how to deal with homologous peptides, and what cutoffs should be considered relevant. This protocol provides step-by-step instructions with necessary recommendations for prediction of the best T cell epitope candidates with the newly developed online tool called TepiTool. TepiTool, which is part of the Immune Epitope Database (IEDB), provides some of the top MHC binding prediction algorithms for number of species including humans, chimpanzees, bovines, gorillas, macaques, mice, and pigs. The TepiTool is freely accessible at http://tools.iedb.org/tepitool/. © 2016 by John Wiley & Sons, Inc.

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

    PubMed Central

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

    1994-01-01

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

  19. Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines.

    PubMed Central

    de Velasco, E A; Merkus, D; Anderton, S; Verheul, A F; Lizzio, E F; Van der Zee, R; Van Eden, W; Hoffman, T; Verhoef, J; Snippe, H

    1995-01-01

    Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed. PMID:7532630

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

    PubMed Central

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

    2014-01-01

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

  1. A synthetic multi-epitope antigen enhances hepatitis C virus-specific B- and T-cell responses.

    PubMed

    Yang, Guimei; Chen, Shuwen; Zhu, Xiangying; Liang, Shenghua; Liu, Longding; Ren, Daming

    2011-04-01

    Combining results from previous studies, a multi-epitope antigen PCXZ against the hepatitis C virus was synthesized in this study. The antigenic specificity of PCXZ was determined by recognizing antibodies in serum samples from hepatitis C virus patients, but not from healthy subjects or subjects who had the hepatitis B virus. The characteristics of PCXZ immunogenicity were evaluated in BALB/c mice. Strong antibody responses were generated in mice immunized with either naked PCXZ or PCXZ in Freund's adjuvant. As for the T-cell responses, Freund's adjuvant significantly increased interferon-γ secretion and enhanced the lytic activity of cytotoxic T lymphocytes. The epitope Pa, one component of PCXZ, made the most significant contribution to specific CTL lysis; this epitope was also a B-cell epitope and was able to induce high IgG titers. In summary, PCXZ was found to be highly immunogenic, and elicited both humoral and cellular immune responses in mice.

  2. A West Nile virus CD4 T cell epitope improves the immunogenicity of dengue virus serotype 2 vaccines.

    PubMed

    Hughes, Holly R; Crill, Wayne D; Davis, Brent S; Chang, Gwong-Jen J

    2012-03-15

    Flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are among the most prevalent human disease-causing arboviruses world-wide. As they continue to expand their geographic range, multivalent flavivirus vaccines may become an important public health tool. Here we describe the immune kinetics of WNV DNA vaccination and the identification of a CD4 epitope that increases heterologous flavivirus vaccine immunogenicity. Lethal WNV challenge two days post-vaccination resulted in 90% protection with complete protection by four days, and was temporally associated with a rapid influx of activated CD4 T cells. CD4 T cells from WNV vaccinated mice could be stimulated from epitopic regions in the envelope protein transmembrane domain. Incorporation of this WNV epitope into DENV-2 DNA and virus-like particle vaccines significantly increased neutralizing antibody titers. Incorporating such potent epitopes into multivalent flavivirus vaccines could improve their immunogenicity and may help alleviate concerns of imbalanced immunity in multivalent vaccine approaches.

  3. A systematic review of T-cell epitopes in hepatitis B virus: identification, genotypic variation and relevance to antiviral therapeutics.

    PubMed

    Desmond, Christopher P; Bartholomeusz, Angeline; Gaudieri, Silvana; Revill, Peter A; Lewin, Sharon R

    2008-01-01

    The immune response to hepatitis B virus (HBV) is important for both viral control and disease pathogenesis. A detailed understanding of the HBV-specific T-cell responses may potentially lead to novel therapeutic strategies for HBV. All English language journal articles (including articles in press) up to October 2007 were retrieved using searches of MEDLINE, EMBASE and the Cochrane Controlled Trial Registry. An extensive database of HBV sequences (SeqHepB) and GenBank were used to assess the degree of sequence variation in each epitope. The new standardized nomenclature for HBV amino acid position number was applied to all previously defined epitopes. Forty-four HBV-specific human leukocyte antigen (HLA) class I restricted and 32 HBV-specific HLA class II restricted epitopes have been defined and have been identified in all HBV genes. The majority of HLA class I restricted epitopes have been defined in HLA-A2-positive individuals in the setting of acute HBV infection. There is significant sequence variation of these epitopes within and between HBV genotypes. Newer HBV immunotherapeutics appear promising but are still in early phases of development. Identification of HBV-specific epitopes in non-HLA-A2-positive individuals and recognition of genotypic variation across epitopes are important for the future development of novel immunotherapeutic strategies for the management of chronic HBV infection.

  4. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

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

    PubMed

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

    2015-01-01

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

  6. The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies.

    PubMed

    Zola, H; Fusco, M; Ridings, J; Flego, L R; Weedon, H M; Nicholson, I; Organ, N; Roberton, D M; Macardle, P J

    1996-11-01

    The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.

  7. T cell memory to evolutionarily conserved and shared hemagglutinin epitopes of H1N1 viruses: a pilot scale study

    PubMed Central

    2013-01-01

    Background The 2009 pandemic influenza was milder than expected. Based on the apparent lack of pre-existing cross-protective antibodies to the A (H1N1)pdm09 strain, it was hypothesized that pre-existing CD4+ T cellular immunity provided the crucial immunity that led to an attenuation of disease severity. We carried out a pilot scale study by conducting in silico and in vitro T cellular assays in healthy population, to evaluate the pre-existing immunity to A (H1N1)pdm09 strain. Methods Large-scale epitope prediction analysis was done by examining the NCBI available (H1N1) HA proteins. NetMHCIIpan, an eptiope prediction tool was used to identify the putative and shared CD4+ T cell epitopes between seasonal H1N1 and A (H1N1)pdm09 strains. To identify the immunogenicity of these putative epitopes, human IFN-γ-ELISPOT assays were conducted using the peripheral blood mononuclear cells from fourteen healthy human donors. All donors were screened for the HLA-DRB1 alleles. Results Epitope-specific CD4+ T cellular memory responses (IFN-γ) were generated to highly conserved HA epitopes from majority of the donors (93%). Higher magnitude of the CD4+ T cell responses was observed in the older adults. The study identified two HA2 immunodominant CD4+ T cell epitopes, of which one was found to be novel. Conclusions The current study provides a compelling evidence of HA epitope specific CD4+ T cellular memory towards A (H1N1)pdm09 strain. These well-characterized epitopes could recruit alternative immunological pathways to overcome the challenge of annual seasonal flu vaccine escape. PMID:23641949

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

    PubMed Central

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

    2011-01-01

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

  9. CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects.

    PubMed

    Slezak, Stefanie L; Bettinotti, Maria; Selleri, Silvia; Adams, Sharon; Marincola, Francesco M; Stroncek, David F

    2007-03-28

    Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized. Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects. CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides. Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.

  10. Identification of linear B-cell epitopes on goose parvovirus non-structural protein.

    PubMed

    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.

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

    PubMed

    Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2012-01-01

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

  12. Integrated computational prediction and experimental validation identifies promiscuous T cell epitopes in the proteome of Mycobacterium bovis

    PubMed Central

    Farrell, Damien; Jones, Gareth; Pirson, Christopher; Malone, Kerri; Rue-Albrecht, Kevin; Chubb, Anthony J.; Vordermeier, Martin

    2016-01-01

    The discovery of novel antigens is an essential requirement in devising new diagnostics or vaccines for use in control programmes against human tuberculosis (TB) and bovine tuberculosis (bTB). Identification of potential epitopes recognised by CD4+ T cells requires prediction of peptide binding to MHC class-II, an obligatory prerequisite for T cell recognition. To comprehensively prioritise potential MHC-II-binding epitopes from Mycobacterium bovis, the agent of bTB and zoonotic TB in humans, we integrated three binding prediction methods with the M. bovisproteome using a subset of human HLA alleles to approximate the binding of epitope-containing peptides to the bovine MHC class II molecule BoLA-DRB3. Two parallel strategies were then applied to filter the resulting set of binders: identification of the top-scoring binders or clusters of binders. Our approach was tested experimentally by assessing the capacity of predicted promiscuous peptides to drive interferon-γ secretion from T cells of M. bovis infected cattle. Thus, 376 20-mer peptides, were synthesised (270 predicted epitopes, 94 random peptides with low predictive scores and 12 positive controls of known epitopes). The results of this validation demonstrated significant enrichment (>24 %) of promiscuously recognised peptides predicted in our selection strategies, compared with randomly selected peptides with low prediction scores. Our strategy offers a general approach to the identification of promiscuous epitopes tailored to target populations where there is limited knowledge of MHC allelic diversity. PMID:28348866

  13. A T-cell epitope on NS3 non-structural protein enhances the B and T cell responses elicited by dendrimeric constructions against CSFV in domestic pigs.

    PubMed

    Tarradas, Joan; Monsó, Marta; Fraile, Lorenzo; de la Torre, Beatriz G; Muñoz, Marta; Rosell, Rosa; Riquelme, Cristina; Pérez, Lester Josué; Nofrarías, Miquel; Domingo, Mariano; Sobrino, Francisco; Andreu, David; Ganges, Llilianne

    2012-11-15

    It has been recently reported by our group that dendrimeric constructs combining B- and T-cell epitopes from classical swine fever virus (CSFV) provided partial protection against experimental infection. This research evaluated four newly designed constructions while taking into account our previous work, including the direct implication that a T-cell epitope from the NS3 protein contributes to the generation of the immune response against CSFV. To this end, the dendrimeric constructions, including either this NS3 T-cell epitope alone or two different B-cell epitopes without this T-cell epitope, were used to immunise pigs. Thus, construct 1, containing the NS3 T-cell epitope and four copies of a previously described B-cell epitope, significantly reduced the clinical scores and RNA viral loads after challenge relative to the control group. In three out of six animals in this group, vaccination achieved partial protection and was associated with IFN-gamma producing-cells and neutralising antibodies. In contrast, the pigs immunised with construct 2, again with four copies of the B epitope of construct 1 but lacking the T-cell motif, developed more severe clinical signs. Finally, the additional constructs 3 and 4 included four copies of a B epitope that was different from the epitope used in constructs 1 and 2 with or without the abovementioned NS3 T-cell epitope, respectively. Pigs immunised with these latter constructs developed low levels of peptide-specific antibodies that correlated with equally low levels of cellular responses, an absence of neutralising antibodies and a lack of protection. Even so, the clinical scores in the first week after the challenge were less severe for animals vaccinated with construct 3 than for those given construct 4. Our results confirm the relevant role of the B-cell epitope in residues 694-712 of the glycoprotein E2 (which is used in both constructs 1 and 2) for protection against CSFV, as well as the appropriateness of the newly

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  16. Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

    PubMed Central

    Cannella, Anthony P.; Arlehamn, Cecilia S. Lindestam; Sidney, John; Patra, Kailash P.; Torres, Katherine; Tsolis, Renee M.; Liang, Li; Felgner, Philip L.; Saito, Mayuko; Gotuzzo, Eduardo; Gilman, Robert H.; Sette, Alessandro

    2014-01-01

    Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen. PMID:24126518

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

    PubMed

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

    2013-04-01

    Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased.

  19. Immunogenicity of recombinant Mycobacterium bovis bacille Calmette–Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens

    PubMed Central

    2013-01-01

    Recombinant Mycobacterium bovis bacille Calmette–Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4+ and CD8+ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB. PMID:23458635

  20. Immunogenicity of recombinant Mycobacterium bovis bacille Calmette-Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens.

    PubMed

    Mohamud, Rohimah; Azlan, Maryam; Yero, Daniel; Alvarez, Nadine; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd-Nor

    2013-01-01

    Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2012-05-01

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

  3. IgE epitope proximity determines immune complex shape and effector cell activation capacity

    PubMed Central

    Gieras, Anna; Linhart, Birgit; Roux, Kenneth H.; Dutta, Moumita; Khodoun, Marat; Zafred, Domen; Cabauatan, Clarissa R.; Lupinek, Christian; Weber, Milena; Focke-Tejkl, Margarete; Keller, Walter; Finkelman, Fred D.; Valenta, Rudolf

    2016-01-01

    Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases. PMID:26684291

  4. Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

    PubMed Central

    da Silva, Andréa N. M. Rangel; Nascimento, Eduardo J. M.; Cordeiro, Marli Tenório; Gil, Laura H. V. G.; Montenegro, Silvia M. L.; Marques, Ernesto T. A.

    2009-01-01

    Background Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. Methodology/Principal Findings Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51–65, 71–90, 131–170, 196–210 and 246–260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51–65), 27 and 28 (aa131–150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51–65, 131–170, 196–210 and 246–260 elicited the highest antibody response and epitopes131–170, 196–210 and 246–260, elicited IFN-γ production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. Conclusions/Significance Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine. PMID:19826631

  5. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses.

    PubMed

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R; Blanco, Esther; Bárcena, Juan

    2016-08-23

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.

  6. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

    PubMed Central

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R.; Blanco, Esther; Bárcena, Juan

    2016-01-01

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge. PMID:27549017

  7. In situ localization of epidermal stem cells using a novel multi epitope ligand cartography approach.

    PubMed

    Ruetze, Martin; Gallinat, Stefan; Wenck, Horst; Deppert, Wolfgang; Knott, Anja

    2010-06-01

    Precise knowledge of the frequency and localization of epidermal stem cells within skin tissue would further our understanding of their role in maintaining skin homeostasis. As a novel approach we used the recently developed method of multi epitope ligand cartography, applying a set of described putative epidermal stem cell markers. Bioinformatic evaluation of the data led to the identification of several discrete basal keratinocyte populations, but none of them displayed the complete stem cell marker set. The distribution of the keratinocyte populations within the tissue was remarkably heterogeneous, but determination of distance relationships revealed a population of quiescent cells highly expressing p63 and the integrins alpha(6)/beta(1) that represent origins of a gradual differentiation lineage. This population comprises about 6% of all basal cells, shows a scattered distribution pattern and could also be found in keratinocyte holoclone colonies. The data suggest that this population identifies interfollicular epidermal stem cells.

  8. CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

    PubMed

    Wu, Te-Lang; Li, Hua; Faust, Susan M; Chi, Emily; Zhou, Shangzhen; Wright, Fraser; High, Katherine A; Ertl, Hildegund C J

    2014-01-01

    Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

  9. Synthetic peptides containing B- and T-cell epitope of dengue virus-2 E domain III provoked B- and T-cell responses.

    PubMed

    Li, Shanfeng; Peng, Liang; Zhao, Wei; Zhong, Hua; Zhang, Fuchun; Yan, Ziqiang; Cao, Hong

    2011-05-09

    Our previous work applied a combination of bioinformatics approaches and in vitro assays to identify the dengue-2 virus (DENV-2)-specific B- and T-cell epitopes. In this report, we first evaluated the antigenicity of both B- and T-cell epitopes reacting with different sera against DENV-2 by ELISA as well as the ability of T-cell epitope to activate CD4(+) T-cell producing IFN-γ using ELISPOT, which showed a specific reactivity between either B- or T-cell epitope and DENV-2 antisera, and a significant increase of IFN-γ producing cells in DENV-2 infected mice. Then, a multi-epitope peptide containing the above B-, T-cell epitopes of envelope domain III (EDIII) of DENV-2 and pan-DR epitope (PADRE) was bioinformatically designed and synthesized. The verification of its immunogenicity and protective effect was performed in in vitro and in vivo experiments. The results showed that a high level of antibody in mice elicited by the multi-epitope peptide was detected by ELISA and the anti-peptide sera binding to the vero cells infected with DEN-2 was observed with immunofluorescence test. More importantly, the peptide could induce lymphoproliferation in vitro and a predominant Th1 type of immune response was examined by flow cytometry. We also found that the virus replication in the mice vaccinated with the multi-epitope peptide was obviously less than that of the control groups. These results may provide some important information for the development of dengue vaccine.

  10. Novel, in-natural-infection subdominant HIV-1 CD8+ T-cell epitopes revealed in human recipients of conserved-region T-cell vaccines.

    PubMed

    Borthwick, Nicola; Lin, Zhansong; Akahoshi, Tomohiro; Llano, Anuska; Silva-Arrieta, Sandra; Ahmed, Tina; Dorrell, Lucy; Brander, Christian; Murakoshi, Hayato; Takiguchi, Masafumi; Hanke, Tomáš

    2017-01-01

    Fine definition of targeted CD8+ T-cell epitopes and their human leucocyte antigen (HLA) class I restriction informs iterative improvements of HIV-1 T-cell vaccine designs and may predict early vaccine success or failure. Here, lymphocytes from volunteers, who had received candidate HIVconsv vaccines expressing conserved sub-protein regions of HIV-1, were used to define the optimum-length target epitopes and their HLA restriction. In HIV-1-positive patients, CD8+ T-cell responses predominantly recognize immunodominant, but hypervariable and therefore less protective epitopes. The less variable, more protective epitopes in conserved regions are typically subdominant. Therefore, induction of strong responses to conserved regions by vaccination provides an opportunity to discover novel important epitopes. Cryopreserved lymphocytes from vaccine recipients were expanded by stimulation with 15-mer responder peptides for 10 days to establish short term-cell-line (STCL) effector cells. These were subjected to intracellular cytokine staining using serially truncated peptides and peptide-pulsed 721.221 cells expressing individual HLA class I alleles to define minimal epitope length and HLA restriction by stimulation of IFN-γ and TNF-α production and surface expression of CD107a. Using lymphocyte samples of 12 vaccine recipients, we defined 14 previously unreported optimal CD8+ T-cell HIV-1 epitopes and their four-digit HLA allele restriction (6 HLA-A, 7 HLA-B and 1 HLA-C alleles). Further 13 novel targets with incomplete information were revealed. The high rate of discovery of novel CD8+ T-cell effector epitopes warrants further epitope mining in recipients of the conserved-region vaccines in other populations and informs development of HIV-1/AIDS vaccines. ClinicalTrials.gov NCT01151319.

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

    PubMed

    Wen, Jinsheng; Duan, Zhiliang; Jiang, Lifang

    2010-04-01

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

  12. Potential T cell epitopes of Mycobacterium tuberculosis that can instigate molecular mimicry against host: implications in autoimmune pathogenesis.

    PubMed

    Chodisetti, Sathi Babu; Rai, Pradeep K; Gowthaman, Uthaman; Pahari, Susanta; Agrewala, Javed N

    2012-03-21

    Molecular mimicry between microbial antigens and host-proteins is one of the etiological enigmas for the occurrence of autoimmune diseases. T cells that recognize cross-reactive epitopes may trigger autoimmune reactions. Intriguingly, autoimmune diseases have been reported to be prevalent in tuberculosis endemic populations. Further, association of Mycobacterium tuberculosis (M. tuberculosis) has been implicated in different autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Although, in silico analyses have identified a number of M. tuberculosis specific vaccine candidates, the analysis on prospective cross-reactive epitopes, that may elicit autoimmune response, has not been yet attempted. Here, we have employed bioinformatics tools to determine T cell epitopes of homologous antigenic regions between M. tuberculosis and human proteomes. Employing bioinformatics tools, we have identified potentially cross-reactive T cell epitopes restricted to predominant class I and II alleles of human leukocyte antigens (HLA). These are similar to peptides of mycobacterial proteins and considerable numbers of them are promiscuous. Some of the identified antigens corroborated with established autoimmune diseases linked with mycobacterial infection. The present study reveals many target proteins and their putative T cell epitopes that might have significant application in understanding the molecular basis of possible T cell autoimmune reactions during M. tuberculosis infections.

  13. Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

    PubMed Central

    Yang, Hui-Jie; Zhang, Jin-Yong; Wei, Chao; Yang, Liu-Yang; Zuo, Qian-Fei; Zhuang, Yuan; Feng, You-Jun; Srinivas, Swaminath; Zeng, Hao; Zou, Quan-Ming

    2016-01-01

    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. PMID:26895191

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

    PubMed

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

    2017-03-13

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

  15. Immunogenicity of epitope vaccines targeting different B cell antigenic determinants of human α-synuclein: feasibility study.

    PubMed

    Ghochikyan, Anahit; Petrushina, Irina; Davtyan, Hayk; Hovakimyan, Armine; Saing, Tommy; Davtyan, Arpine; Cribbs, David H; Agadjanyan, Michael G

    2014-02-07

    Immunotherapeutic approaches reducing α-synuclein deposits may provide therapeutic benefit for Dementia with Lewy Bodies (DLB). Immunization with full-length human α-synuclein (hα-Syn) protein in a Parkinson's disease mouse model decreased the accumulation of the aggregated forms of this protein in neurons and reduced neurodegeneration. To enhance the immunogenicity of candidate vaccines and to avoid the risk of autoreactive anti-hα-Syn T-helper (Th) cell responses, we generated three peptide-based epitope vaccines composed of different B-cell epitopes of hα-Syn fused with a "non-self" Th epitope from tetanus toxin (P30). Immunization of mice with these epitope vaccines produced high titers of anti-hα-Syn antibodies that bound to Lewy bodies (LBs) and Lewy neurites (LNs) in brain tissue from DLB cases and induced robust Th cell responses to P30, but not to hα-Syn. Further development of these first generation epitope vaccines may facilitate induction of anti-hα-Syn immunotherapy without producing potentially harmful autoreactive Th cell responses.

  16. Identification of Caucasian CD4 T cell epitopes on the circumsporozoite protein of Plasmodium vivax. T cell memory.

    PubMed

    Bilsborough, J; Carlisle, M; Good, M F

    1993-07-15

    We have identified a population of Caucasians with a defined past history of infection with Plasmodium vivax malaria. Using purified synthetic peptides overlapping the sequence of the circumsporozoite protein, we determined the percentage of individuals whose T cells proliferated or secreted IFN-gamma in response to peptide stimulation, for both this population and a population of nonmalaria-exposed control individuals. A number of peptides were recognized by both groups, but 11 peptides were uniquely recognized by the exposed population, and thus represented malaria-specific T cell epitopes. CD4 T cells were found to be responsible for the proliferative response. Humans last exposed to vivax sporozoites as long ago as 49 yr responded as well or better to these malaria-specific epitopes as individuals exposed within the previous month. Since such malaria-induced memory response may not be a feature of Plasmodium falciparum infections, and since P. falciparum does not have a persisting hypnozoite stage, our data argue that the persistence of T cell memory to vivax epitopes may result from antigenic persistence in the liver.

  17. Apoptotic Epitope-Specific CD8+ T Cells and Interferon Signaling Intersect in Chronic Hepatitis C Virus Infection.

    PubMed

    Martini, Helene; Citro, Alessandra; Martire, Carmela; D'Ettorre, Gabriella; Labbadia, Giancarlo; Accapezzato, Daniele; Piconese, Silvia; De Marzio, Paolo; Cavallari, Eugenio N; Calvo, Ludovica; Rizzo, Fabiana; Severa, Martina; Coccia, Eliana M; Grazi, Gian Luca; Di Filippo, Simona; Sidney, John; Vullo, Vincenzo; Sette, Alessandro; Barnaba, Vincenzo

    2016-02-15

    CD8(+) T cells specific to caspase-cleaved antigens derived from apoptotic T cells represent a principal player in chronic immune activation. Here, we found that both apoptotic epitope-specific and hepatitis C virus (HCV)-specific CD8(+) T cells were mostly confined within the effector memory (EM) or terminally differentiated EM CD45RA(+) cell subsets expressing a dysfunctional T-helper 1-like signature program in chronic HCV infection. However, apoptotic epitope-specific CD8(+) T cells produced tumor necrosis factor α and interleukin 2 at the intrahepatic level significantly more than HCV-specific CD8(+) T cells, despite both populations expressing high levels of programmed death 1 receptor. Contextually, only apoptotic epitope-specific CD8(+) T cells correlated with both interferon-stimulated gene levels in T cells and hepatic fibrosis score. Together, these data suggest that, compared with HCV-specific CD8(+) T cells, apoptotic epitope-specific CD8(+) T cells can better sustain chronic immune activation, owing to their capacity to produce tumor necrosis factor α, and exhibit greater resistance to inhibitory signals during chronic HCV infection.

  18. T-cell epitope analysis of Mag 3, an important allergen from the house dust mite, Dermatophagoides farinae.

    PubMed

    Kawamoto, S; Ohno, K; Tategaki, A; Aki, T; Shigeta, S; Jyo, T; Suzuki, O; Ono, K

    2000-04-03

    Here we describe the detection of T-cell epitope region on the house dust mite allergen Mag 3, which has been shown to trigger T-cell proliferation in mite-allergic asthmatic patients. We first examined murine T-cell epitope using T-cell fraction prepared from recombinant Mag 3 (r-Mag 3)-primed H-2k mice. Initial proliferation assay with truncated r-Mag 3 indicated that N-terminal 113 amino acid region was required for triggering T-cell activation. Subsequent epitope scanning with synthetic overlapping peptides revealed that T-cell reactive region was assigned within amino acid range 56-75. We also explored human T-cell determinant using specific T-cells from mite-allergic patients. Intriguingly, we found that amino acid range 56-85, a portion partially overlapping with that identified in r-Mag 3-primed mice, was exclusively recognized by T-cells from different patients. Further investigation of unique T-cell epitope region found in this study would provide insight into the development of animal therapeutic model and/or peptide vaccine for asthma.

  19. Glycoproteomic characterization of carriers of the CD15/Lewisx epitope on Hodgkin's Reed-Sternberg cells.

    PubMed

    Powlesland, Alex S; Barrio, Maria Marcela; Mordoh, José; Hitchen, Paul G; Dell, Anne; Drickamer, Kurt; Taylor, Maureen E

    2011-03-24

    The Lewisx trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewisx structures on Hodgkin's Reed-Sternberg cells have not been identified. GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewisx and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines. Multiple glycoproteins that bind to GalMBP and carry CD15/Lewisx have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin's Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewisx-bearing glycoproteins. CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewisx on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.

  20. Multiple linear epitopes (B-cell, CTL and Th) of JEV expressed in recombinant MVA as multiple epitope vaccine induces a protective immune response.

    PubMed

    Wang, Fengjuan; Feng, Xiuli; Zheng, Qisheng; Hou, Hongyan; Cao, Ruibing; Zhou, Bin; Liu, Qingtao; Liu, Xiaodong; Pang, Ran; Zhao, Jin; Deng, Wenlei; Chen, Puyan

    2012-09-17

    Epitope-based vaccination might play an important role in the protective immunity against Japanese encephalitis virus (JEV) infection. The purpose of the study is to evaluate the immune characteristics of recombinant MVA carrying multi-epitope gene of JEV (rMVA-mep). The synthetic gene containing critical epitopes (B-cell, CTL and Th) of JEV was cloned into the eukaryotic expression vector pGEM-K1L, and the rMVA-mep was prepared. BALB/c mice were immunized with different dosages of purified rMVA-mep and the immune responses were determined in the form of protective response against JEV, antibodies titers (IgG1 and IgG2a), spleen cell lymphocyte proliferation, and the levels of interferon-γ and interleukin-4 cytokines. The results showed that live rMVA-mep elicited strongly immune responses in dose-dependent manner, and the highest level of immune responses was observed from the groups immunized with 107 TCID50 rMVA-mep among the experimental three concentrations. There were almost no difference of cytokines and neutralizing antibody titers among 107 TCID50 rMVA-mep, recombinant ED3 and inactivated JEV vaccine. It was noteworthy that rMVA-mep vaccination potentiates the Th1 and Th2-type immune responses in dose-dependent manner, and was sufficient to protect the mice survival against lethal JEV challenge. These findings demonstrated that rMVA-mep can produce adequate humoral and cellular immune responses, and protection in mice, which suggested that rMVA-mep might be an attractive candidate vaccine for preventing JEV infection.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Nadugala, Mahesha N.

    2016-01-01

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

  3. Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

    PubMed Central

    Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

    2016-01-01

    This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

  4. Similar Responses of Intestinal T Cells From Untreated Children and Adults With Celiac Disease to Deamidated Gluten Epitopes.

    PubMed

    Ráki, Melinda; Dahal-Koirala, Shiva; Yu, Hao; Korponay-Szabó, Ilma R; Gyimesi, Judit; Castillejo, Gemma; Jahnsen, Jørgen; Qiao, Shuo-Wang; Sollid, Ludvig M

    2017-09-01

    Celiac disease is a chronic small intestinal inflammatory disorder mediated by an immune response to gluten peptides in genetically susceptible individuals. Celiac disease is often diagnosed in early childhood, but some patients receive a diagnosis late in life. It is uncertain whether pediatric celiac disease is distinct from adult celiac disease. It has been proposed that gluten-reactive T cells in children recognize deamidated and native gluten epitopes, whereas T cells from adults only recognize deamidated gluten peptides. We studied the repertoire of gluten epitopes recognized by T cells from children and adults. We examined T-cell responses against gluten by generating T-cell lines and T-cell clones from intestinal biopsies of adults and children and tested proliferative response to various gluten peptides. We analyzed T cells from 14 children (2-5 years old) at high risk for celiac disease who were followed for celiac disease development. We also analyzed T cells from 6 adults (26-55 years old) with untreated celiac disease. All children and adults were positive for HLA-DQ2.5. Biopsies were incubated with gluten digested with chymotrypsin (modified or unmodified by the enzyme transglutaminase 2) or the peptic-tryptic digest of gliadin (in native and deamidated forms) before T-cell collection. Levels of T-cell responses were higher to deamidated gluten than to native gluten in children and adults. T cells from children and adults each reacted to multiple gluten epitopes. Several T-cell clones were cross-reactive, especially clones that recognized epitopes from γ-and ω-gliadin. About half of the generated T-cell clones from children and adults reacted to unknown epitopes. T-cell responses to different gluten peptides appear to be similar between adults and children at the time of diagnosis of celiac disease. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-02-01

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

  6. Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion

    PubMed Central

    Ameres, Stefanie; Mautner, Josef; Schlott, Fabian; Neuenhahn, Michael; Busch, Dirk H.; Plachter, Bodo; Moosmann, Andreas

    2013-01-01

    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. PMID:23717207

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

    PubMed

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

    2014-03-26

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

  8. Antibodies to two ZP3 B cell epitopes affect zona pellucida assembly.

    PubMed

    Borillo, Jason; Coonrod, Scott A; Wu, Jean; Zhou, Cindy; Lou, Yahuan

    2008-07-01

    Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335-342) and ZP3 (171-180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.

  9. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

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

    2014-01-01

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

  10. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products

    PubMed Central

    1990-01-01

    A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well- characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as

  11. [Improved weighted cross-correlation coefficient with a new kernel and its application in predicting T cell epitopes].

    PubMed

    Huang, Jing; Ma, Jian-hua; Liu, Nan; Qian, Shan-shan

    2010-10-01

    We designed a weighted cross-correlation coefficient considering the "anchor" of the T cell epitopes, and used an evolutionary algorithm to search for an optimal weight vector. A SVM model with this new peptide similarity kernel was evaluated on a T-cell data set. The results demonstrated a good performance of this method.

  12. Targeting of Conserved Gag-Epitopes in Early HIV Infection Is Associated with Lower Plasma Viral Load and Slower CD4+ T Cell Depletion

    PubMed Central

    Perez, Carina L.; Milush, Jeffrey M.; Buggert, Marcus; Eriksson, Emily M.; Larsen, Mette V.; Liegler, Teri; Hartogensis, Wendy; Bacchetti, Peter; Lund, Ole; Hecht, Frederick M.; Nixon, Douglas F.

    2013-01-01

    Abstract We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8+ T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8+ T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8+ T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log10 difference). Furthermore, the rate of CD4+ T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome. PMID:23140171

  13. Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.

    PubMed Central

    Mozes, E; Dayan, M; Zisman, E; Brocke, S; Licht, A; Pecht, I

    1989-01-01

    MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC. PMID:2480232

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

    PubMed

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

    2015-05-01

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

  15. Optimization of therapeutic proteins to delete T-cell epitopes while maintaining beneficial residue interactions.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-04-01

    Exogenous enzymes, signaling peptides, and other classes of nonhuman proteins represent a potentially massive but largely untapped pool of biotherapeutic agents. Adapting a foreign protein for therapeutic use poses numerous design challenges. We focus here on one significant problem: modifying the protein to mitigate the immune response mounted against "non-self" proteins, while not adversely affecting the protein's stability or therapeutic activity. In order to propose such variants suitable for experimental evaluation, this paper develops a computational method to select sets of mutations predicted to delete immunogenic T-cell epitopes, as evaluated by a 9-mer potential, while simultaneously maintaining important residues and residue interactions, as evaluated by one- and two-body potentials. While this design problem is NP-hard, we develop an integer programming approach that works very well in practice. We demonstrate the effectiveness of our approach by developing plans for biotherapeutic proteins that, in previous studies, have been partially deimmunized via extensive experimental characterization and modification of limited segments. In contrast, our global optimization technique considers an entire protein and accounts for all residues, residue interactions, and epitopes in proposing candidates worth subjecting to experimental evaluation.

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

    PubMed Central

    1996-01-01

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

  17. Type III hypersensitivity reactions to a B cell epitope antigen are abrogated using a depot forming vaccine platform.

    PubMed

    MacDonald, Lisa D; MacKay, Alecia; Kaliaperumal, Valarmathy; Weir, Genevieve; Penwell, Andrea; Rajagopalan, Rajkannan; Langley, Joanne; Halperin, Scott; Mansour, Marc; Stanford, Marianne M

    2017-09-21

    Peptide antigens are combined with an adjuvant in order to increase immunogenicity in vivo. The immunogenicity and safety of a RSV vaccine formulated in a novel oil-based platform, DepoVax™ (DPX), was compared to an alum formulation. A peptide B cell epitope derived from RSV small hydrophobic ectodomain (SHe) served as the antigen. Both vaccines induced SHe-specific antibodies after immunization of mice. A single dose of the DPX-based formulation resulted in anti-SHe titres for up to 20 weeks. Boosting with Alum-SHe, but not with DPX-SHe, led to unexpected clinical signs such as decreased activity, cyanosis and drop in body temperature in mice but not in rabbits. The severity of adverse reactions correlated with magnitude of SHe-specific IgG immune responses and decreased complement component 3 plasma levels, indicating a type III hypersensitivity reaction. By RP-HPLC analysis, we found that only 8-20% of the antigen was found to be adsorbed to alum in vitro, indicating that this antigen is likely released systemically upon injection in vivo. Clinical signs were not observed in rabbits, indicating the response correlates with peptide dose relative to size of animal. These results suggest that peptide antigens targeted to produce B cell mediated response may result in increased incidence of type III hypersensitivity reactions when delivered in non-depot forming vaccines. The DPX formulation induced strong antibody titres to the antigen without causing adverse events, likely due to the strength of the depot in vivo, and demonstrates the potential safety and immunogenicity of this platform for B cell peptide antigens.

  18. T cell epitopes of insulin defined in HLA-DR4 transgenic mice are derived from preproinsulin and proinsulin

    PubMed Central

    Congia, Mauro; Patel, Salil; Cope, Andrew P.; De Virgiliis, Stefano; Sønderstrup, Grete

    1998-01-01

    Approximately one-half of Caucasians with newly diagnosed insulin-dependent diabetes mellitus (IDDM) have autoantibodies to insulin, and the majority of those express the HLA-DR4 genotype [Ziegler, R., Alper, C. A., Awdeh, Z. L., Castano, L., Brink, S. J., Soeldner, J. S., Jackson, R. A. & Eisenbarth, G. S. (1991) Diabetes 40, 709–714]. However, it has been difficult to demonstrate T cell proliferative responses to human insulin in IDDM patients [Durinovic-Bello, I., Hummel, M. & Ziegler, A. G. (1996) Diabetes 45, 795–800]. We have immunized transgenic mice expressing the susceptible HLA-DR (α1*0101,β1*0401) (hereafter called DRB1*0401) and human CD4 molecules on a murine major histocompatibility complex class II null background, with human preproinsulin (PPI), proinsulin (PI), and insulin and derived large panels of T cell hybridomas to determine the immunogenic epitopes of these proteins. These results show that the prohormones PI or PPI carry the major immunogenic T cell epitope in the DRB1*0401 transgenic mice. The PPI/PI immunodominant epitope LALEGSLQK was localized at the C-peptide/A-chain junction. This T cell epitope PPI/PI LALEGSLQK is unusual because, normally, it is proteolytically destroyed during the maturation of the insulin molecule. Additionally, this T cell epitope is both processed and presented by human DRB1*0401-positive Epstein–Barr virus transformed B cells, and it can also stimulate T cells from the peripheral blood of HLA-DR4-positive patients with type 1 diabetes. These findings may partly explain why susceptibility to type 1 diabetes is associated with HLA-DR4-positive individuals and why T cell responses to the mature insulin protein are rarely detected in IDDM patients. PMID:9520453

  19. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia

    PubMed Central

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-01-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. PMID:22486311

  20. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

    PubMed

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-07-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  1. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Epitopes rationally selected through computational analyses induce T-cell proliferation in mice and are recognized by serum from individuals infected with Schistosoma mansoni.

    PubMed

    Lopes, Marcelo D; Oliveira, Flávio M; Coelho, Ivan E V; Passos, Maria J F; Alves, Clarice C; Taranto, Alex G; Júnior, Moacyr C; Santos, Luciana L; Fonseca, Cristina T; Villar, José A F P; Lopes, Débora O

    2017-04-03

    Schistosomiasis is the second leading cause of death due to parasitic diseases in the world. Seeking an alternative for the control of disease, the World Health Organization funded the genome sequencing of the major species related to schistosomiasis to identify potential vaccines and therapeutic targets. Therefore, the aim of this work was to select T and B-cell epitopes from Schistosoma mansoni through computational analyses and evaluate the immunological potential of epitopes in vitro. Extracellular regions of membrane proteins from the Schistosoma mansoni were used to predict promiscuous epitopes with affinity to different human Major Histocompatibility Class II (MHCII) molecules by bioinformatics analysis. The three-dimensional structure of selected epitopes was constructed and used in molecular docking to verify the interaction with murine MHCII H2-IA(b) . In this process, four epitopes were selected and synthesized to assess their ability to stimulate proliferation of CD4(+) T lymphocytes in mice splenocyte cultures. The results showed that Sm041370 and Sm168240 epitopes induced significant cell proliferation. Additionally, the four epitopes were used as antigens in the Indirect Enzyme-Linked Immunosorbent Assay (ELISA) to assess the recognition by serum from individuals infected with Schistosoma mansoni. Sm140560, Sm168240 and Sm041370 epitopes were recognized by infected individuals IgG antibodies. Therefore, Sm041370 and Sm168240 epitopes that stood out in in silico and in vitro analyses could be promising antigens in schistosomiasis vaccine development or diagnostic kits. This article is protected by copyright. All rights reserved.

  5. Hypoxia upregulates the expression of the O-linked N-acetylglucosamine containing epitope H in human ependymal cells.

    PubMed

    Arvanitis, Leonidas D; Vassiou, Katerina; Kotrotsios, Anastasios; Sgantzos, Markos N

    2011-02-15

    Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by mouse IgM monoclonal antibody H (mabH). Epitope H is present in several types of cells and in several polypeptides outside the CNS. Previous results have shown that in the adult human brains, epitope H is confined mostly to a minority of fibrous astrocytes, and it is greatly upregulated in the reactive astrocytes. Post-translational modification with O-GlcNAc occurs on many proteins involved in several cell processes, such as cell cycle progression, apoptosis, proteasome degradation pathways, and modulation of cellular function in response to nutrition and stress. Hypoxia is one of the major causes of cellular stress. Therefore, in this study, we used the mAbH and the indirect immunoperoxidase method to investigate the expression of epitope H in ependymal cells in brains of persons who died with signs of hypoxic encephalopathy. The results of the present study showed that practically all ependymal cells showed cytoplasmic staining for epitope H in supranuclear cytoplasm in the brain of two premature neonates and in ten infants who died with signs of hypoxic encephalopathy. However, the overwhelming majority of ependymal cells of the nine human embryos taken from legal abortions, ranging from 26 days until 13 weeks of gestational age, and of the ten infants' brains without any sign of hypoxic encephalopathy remained negative. Only occasionally did the ependymal cells show weak cytoplasmic staining in some foci. In addition, the reactive astrocytes in the hypoxic brains showed strong cytoplasmic staining, confirming previous results.

  6. Loss of multi-epitope specificity in memory CD4(+) T cell responses to B. pertussis with age.

    PubMed

    Han, Wanda G H; van Twillert, Inonge; Poelen, Martien C M; Helm, Kina; van de Kassteele, Jan; Verheij, Theo J M; Versteegh, Florens G A; Boog, Claire J P; van Els, Cécile A C M

    2013-01-01

    Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4(+) T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4(+) T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4(+) T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.

  7. Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells.

    USDA-ARS?s Scientific Manuscript database

    To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found...

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

    PubMed

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

    1996-07-01

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

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

    PubMed Central

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

    2014-01-01

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

  10. Anti-human embryonic stem cell monoclonal antibody Hesca-2 binds to a glycan epitope commonly found on carcinomas.

    PubMed

    Shoreibah, Mohamed G; Jackson, Crystal L; Price, Paul W; Meagher, Richard; Godwin, Andrew K; Cai, Qi; Gildersleeve, Jeffrey C

    2011-03-01

    Hesca-2, a monoclonal antibody (mAb) IgM raised to the human embryonic stem cell (hESC) line BG-01v, binds with high affinity (nM) to the disaccharide epitope (Galβ1-3GlcNAc) on a glycan microarray. This epitope was expressed on pluripotent progenitor hESCs in culture, but not in various differentiated cells derived from hESC based on immunofluorescence microscopy. Hesca-2 stains a limited subset of cells in adult human tissues (eg, esophagus and breast). This mAb also crossreacts in immunofluorescence microscopy studies with several human ovarian cancer cell lines and is cytotoxic to them based on the release of cytosolic enzyme lactate dehydrogenase into the media. Hesca-2 immunohistochemically stained tissue from a number of human tumors, including ovary, breast, colon, and esophageal cancer. These data suggest that Hesca-2 recognizes a surface marker found both in stem cells and certain cancer cells.

  11. Human CTLs to wild-type and enhanced epitopes of a novel prostate and breast tumor-associated protein, TARP, lyse human breast cancer cells.

    PubMed

    Oh, SangKon; Terabe, Masaki; Pendleton, C David; Bhattacharyya, Anu; Bera, Tapan K; Epel, Malka; Reiter, Yoram; Phillips, John; Linehan, W Marston; Kasten-Sportes, Claude; Pastan, Ira; Berzofsky, Jay A

    2004-04-01

    Vaccine therapy for prostate and breast cancer may have potential for treating these major causes of death in males and females, respectively. Critical to the development of tumor-specific vaccines is finding and characterizing novel antigens to be recognized by CD8(+) T cells. To define new CD8(+) T-cell tumor antigens, we determined two wild-type HLA-A2 epitopes from a recently found tumor-associated protein, TARP (T-cell receptor gamma alternate reading frame protein), expressed in prostate and breast cancer cells. We were also able to engineer epitope-enhanced peptides by sequence modifications. Both wild-type and enhanced epitopes induced peptide-specific CD8(+) T-cell responses in A2K(b) transgenic mice. In vitro restimulation of human CD8(+) T cells from a prostate cancer patient resulted in CD8(+) T cells reactive to the peptide epitopes that could lyse HLA-A2(+) human breast cancer cells (MCF-7) expressing TARP. Epitope-specific human CD8(+) T cells were also enumerated in patients' peripheral blood by tetramer staining. Our data suggest that HLA-A2-binding TARP epitopes and enhanced epitopes discovered in this study could be incorporated into a potential vaccine for both breast and prostate cancer.

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

    PubMed

    Fikes, John D; Sette, Alessandro

    2003-09-01

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

  13. Identification of bovine T-cell epitopes for three Mycobacterium bovis antigens: MPB70, 19,000 MW and MPB57.

    PubMed Central

    Pollock, J M; Douglas, A J; Mackie, D P; Neill, S D

    1994-01-01

    Bovine tuberculosis remains a serious problem in several regions, partly due to a lack of specific diagnostic tests. The aim of this study was to identify bovine T-cell epitopes for defined Mycobacterium bovis antigens using an experimental model of the natural disease. Panels of synthetic peptides (16-mers with five residue overlaps) were produced from published amino acid sequences for MPB70, the 19,000 MW antigen and MPB57. In vitro lymphocyte proliferation assays were used to identify T-cell epitopes. Lymphocytes from experimentally infected cattle proliferated in response to five epitopes (residues 88-105 and 144-163 for MPB70; 1-16 and 67-84 for the 19,000 MW antigen; and 85-100 for MBP57). These epitopes were not recognized by control, non-infected animals, but were recognized by field reactors to intradermal tuberculin testing. All five epitopes were recognized by three different breeds of cattle (Friesian, Charolais and Simmental). In addition, the bovine T-cell epitopes identified for the 19,000 MW antigen in this study were similar to epitopes previously reported for man and mouse. Thus, as well as identifying candidate reagents for improved diagnostic tests and vaccination, this study provides evidence for genetic promiscuity T-cell recognition of major myobacterial epitopes. PMID:7519175

  14. Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

    PubMed

    Hui, Daniel J; Basner-Tschakarjan, Etiena; Chen, Yifeng; Davidson, Robert J; Buchlis, George; Yazicioglu, Mustafa; Pien, Gary C; Finn, Jonathan D; Haurigot, Virginia; Tai, Alex; Scott, David W; Cousens, Leslie P; Zhou, Shangzhen; De Groot, Anne S; Mingozzi, Federico

    2013-09-01

    Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

  15. Prophylactic immunotherapy of Alzheimer's disease using recombinant amyloid-β B-cell epitope chimeric protein as subunit vaccine.

    PubMed

    Yu, Yun-Zhou; Xu, Qing

    2016-11-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. The neuropathological features of the disease include senile plaques (SPs), neurofibrillary tangles (NFTs) and neuronal loss in affected brain regions. The amyloid cascade hypothesis suggests that production and accumulation of excessive amyloid-β (Aβ) may be the main cause in the onset and progression of Alzheimer's disease. Active and passive immunotherapy targeting Aβ may be the most promising strategy to prevent or treat AD. This commentary focuses on the prophylactic immunotherapy of Alzheimer's disease using recombinant Aβ B-cell epitope chimeric protein as subunit vaccine targeting amyloid-β. We discuss the efficiency and perspective of this type of recombinant subunit protein vaccine and suggest a novel direction on the path to a successful AD immunotherapy. This novel chimeric protein immunogen as subunit vaccine of AD may be designed to mimic the assembly states of Aβ42 or oligomers using multivalent foldable Aβ1-15 (B cell epitopes of Aβ42) and foreign T helper (Th) epitopes (as the T cell epitopes of Aβ42) constructs.

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

    PubMed

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

    2004-09-01

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

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

    PubMed

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

    2016-12-26

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

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

    PubMed

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

    1994-06-15

    Glanzmann's thrombasthenia is an inherited bleeding disorder that results from a deficit of glycoprotein (GP) IIb-IIIa complexes in platelets. Patient (EBV) is an adult male with GP IIb-IIIa levels < 5% of normal values and a history of blood transfusions. Western-blot analysis revealed a strong IgG antibody to GP IIIa in his plasma. The determinants were localized to the minimum-sized fragment of GP IIIa (50 kDa) retained on chymotrypsin-treated platelets and were lost on reduction of disulphides. A female patient (AF), previously described by us [Jallu, V., Pico, M., Chevaleyre, J., Vezon, G., Kunicki, T.J. & Nurden, A.T. (1992) Hum. Antibod. Hybridomas 3, 93-106] developed her anti-GP-IIIa antibody during pregnancy. This antibody was poorly reactive with the 50-kDa proteolytic fragment, yet bound to 115-kDa and 60-kDa hydrolytic products of GP IIIa. Antibodies from both patients recognized the GP-IIIa-like protein of endothelial cells, thus confirming that they were directed against the integrin beta 3-subunit. The (EBV) antibody reacted strongly with GP IIb-IIIa in an antigen capture assay performed with each of a panel of four murine monoclonal antibodies (mAbs) recognizing different epitopes on GP IIb-IIIa. In contrast, that from (AF) was specifically inhibited by AP-3, a murine mAb whose epitope is thought to be localized between amino acids 324-422 of GP IIIa. The residual GP IIb and GP IIIa contents of platelets from each patient were assessed in Western blotting using chemiluminescence detection. SZ-22, a murine mAb to the GP IIb heavy chain (140 kDa), located small amounts of a 130-kDa protein in (EBV) platelets. The anti-GP IIIa mAbs XII F9, P 37 and P 97 revealed trace amounts of protein with a relative mobility identical to that of GP IIIa in both (AF) and (EBV) platelets. This residual GP IIIa represented less than 0.5% of the amount in normal platelets. When, for each patient, plasma was tested in Western blotting against their own platelets

  19. Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development.

    PubMed

    Kovjazin, Riva; Volovitz, Ilan; Daon, Yair; Vider-Shalit, Tal; Azran, Roy; Tsaban, Lea; Carmon, Lior; Louzoun, Yoram

    2011-04-01

    Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.

  20. Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation

    PubMed Central

    1992-01-01

    Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine

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

    PubMed

    Smith, Heath A; McNeel, Douglas G

    2011-10-01

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

  2. Benchmarking B-cell epitope prediction with quantitative dose-response data on antipeptide antibodies: towards novel pharmaceutical product development.

    PubMed

    Caoili, Salvador Eugenio C

    2014-01-01

    B-cell epitope prediction can enable novel pharmaceutical product development. However, a mechanistically framed consensus has yet to emerge on benchmarking such prediction, thus presenting an opportunity to establish standards of practice that circumvent epistemic inconsistencies of casting the epitope prediction task as a binary-classification problem. As an alternative to conventional dichotomous qualitative benchmark data, quantitative dose-response data on antibody-mediated biological effects are more meaningful from an information-theoretic perspective in the sense that such effects may be expressed as probabilities (e.g., of functional inhibition by antibody) for which the Shannon information entropy (SIE) can be evaluated as a measure of informativeness. Accordingly, half-maximal biological effects (e.g., at median inhibitory concentrations of antibody) correspond to maximally informative data while undetectable and maximal biological effects correspond to minimally informative data. This applies to benchmarking B-cell epitope prediction for the design of peptide-based immunogens that elicit antipeptide antibodies with functionally relevant cross-reactivity. Presently, the Immune Epitope Database (IEDB) contains relatively few quantitative dose-response data on such cross-reactivity. Only a small fraction of these IEDB data is maximally informative, and many more of them are minimally informative (i.e., with zero SIE). Nevertheless, the numerous qualitative data in IEDB suggest how to overcome the paucity of informative benchmark data.

  3. Benchmarking B-Cell Epitope Prediction with Quantitative Dose-Response Data on Antipeptide Antibodies: Towards Novel Pharmaceutical Product Development

    PubMed Central

    Caoili, Salvador Eugenio C.

    2014-01-01

    B-cell epitope prediction can enable novel pharmaceutical product development. However, a mechanistically framed consensus has yet to emerge on benchmarking such prediction, thus presenting an opportunity to establish standards of practice that circumvent epistemic inconsistencies of casting the epitope prediction task as a binary-classification problem. As an alternative to conventional dichotomous qualitative benchmark data, quantitative dose-response data on antibody-mediated biological effects are more meaningful from an information-theoretic perspective in the sense that such effects may be expressed as probabilities (e.g., of functional inhibition by antibody) for which the Shannon information entropy (SIE) can be evaluated as a measure of informativeness. Accordingly, half-maximal biological effects (e.g., at median inhibitory concentrations of antibody) correspond to maximally informative data while undetectable and maximal biological effects correspond to minimally informative data. This applies to benchmarking B-cell epitope prediction for the design of peptide-based immunogens that elicit antipeptide antibodies with functionally relevant cross-reactivity. Presently, the Immune Epitope Database (IEDB) contains relatively few quantitative dose-response data on such cross-reactivity. Only a small fraction of these IEDB data is maximally informative, and many more of them are minimally informative (i.e., with zero SIE). Nevertheless, the numerous qualitative data in IEDB suggest how to overcome the paucity of informative benchmark data. PMID:24949474

  4. Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity

    PubMed Central

    Yagita, Yuichi; Kuse, Nozomi; Kuroki, Kimiko; Gatanaga, Hiroyuki; Carlson, Jonathan M.; Chikata, Takayuki; Brumme, Zabrina L.; Murakoshi, Hayato; Akahoshi, Tomohiro; Pfeifer, Nico; Mallal, Simon; John, Mina; Ose, Toyoyuki; Matsubara, Haruki; Kanda, Ryo; Fukunaga, Yuko; Honda, Kazutaka; Kawashima, Yuka; Ariumi, Yasuo; Oka, Shinichi; Maenaka, Katsumi

    2013-01-01

    Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication. PMID:23236061

  5. Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

    PubMed Central

    Sepulveda, P; Lopez-Ribot, J L; Gozalbo, D; Cervera, A; Martinez, J P; Chaffin, W L

    1996-01-01

    We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures. PMID:8926122

  6. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31.

    PubMed

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; Ryu, Chun Jeih

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

  7. A bivalent dendrimeric peptide bearing a T-cell epitope from foot-and-mouth disease virus protein 3A improves humoral response against classical swine fever virus.

    PubMed

    Bohórquez, José Alejandro; Defaus, Sira; Muñoz-González, Sara; Perez-Simó, Marta; Rosell, Rosa; Fraile, Lorenzo; Sobrino, Francisco; Andreu, David; Ganges, Llilianne

    2017-06-15

    Three dendrimeric peptides were synthesized in order to evaluate their immunogenicity and their potential protection against classical swine fever virus (CSFV) in domestic pigs. Construct 1, an optimized version of a previously used dendrimer, had four copies of a B-cell epitope derived from CSFV E2 glycoprotein connected to an also CSFV-derived T-cell epitope through maleimide instead of thioether linkages. Construct 2 was similarly built but included only two copies of the B-cell epitope, and in also bivalent construct 3 the CSFV T-cell epitope was replaced by a previously described one from the 3A protein of foot-and-mouth disease virus (FMDV). Animals were inoculated twice with a 21-day interval and challenged 15days after the second immunization. Clinical signs were recorded daily and ELISA tests were performed to detect antibodies against specific peptide and E2. The neutralising antibody response was assessed 13days after challenge. Despite the change to maleimide connectivity, only partial protection against CSFV was again observed. The best clinical protection was observed in group 3. Animals inoculated with constructs 2 and 3 showed higher anti-peptide humoral response, suggesting that two copies of the B-cell epitope are sufficient or even better than four copies for swine immune recognition. In addition, for construct 3 higher neutralizing antibody titres against CSFV were detected. Our results support the immunogenicity of the CSFV B-cell epitope and the cooperative role of the FMDV 3A T-cell epitope in inducing a neutralising response against CSFV in domestic pigs. This is also the first time that the FMDV T-cell epitope shows effectivity in improving swine immune response against a different virus. Our findings highlight the relevance of dendrimeric peptides as a powerful tool for epitope characterization and antiviral strategies development. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Using injectoporation to deliver genes to mechanosensory hair cells.

    PubMed

    Xiong, Wei; Wagner, Thomas; Yan, Linxuan; Grillet, Nicolas; Müller, Ulrich

    2014-10-01

    Mechanosensation, the transduction of mechanical force into electrochemical signals, allows organisms to detect touch and sound, to register movement and gravity, and to sense changes in cell volume and shape. The hair cells of the mammalian inner ear are the mechanosensors for the detection of sound and head movement. The analysis of gene function in hair cells has been hampered by the lack of an efficient gene transfer method. Here we describe a method termed injectoporation that combines tissue microinjection with electroporation to express cDNAs and shRNAs in mouse cochlear hair cells. Injectoporation allows for gene transfer into dozens of hair cells, and it is compatible with the analysis of hair cell function using imaging approaches and electrophysiology. Tissue dissection and injectoporation can be carried out within a few hours, and the tissue can be cultured for days for subsequent functional analyses.

  9. Coupling of aggregation and immunogenicity in biotherapeutics: T- and B-cell immune epitopes may contain aggregation-prone regions.

    PubMed

    Kumar, Sandeep; Singh, Satish K; Wang, Xiaoling; Rup, Bonita; Gill, Davinder

    2011-05-01

    Biotherapeutics, including recombinant or plasma-derived human proteins and antibody-based molecules, have emerged as an important class of pharmaceuticals. Aggregation and immunogenicity are among the major bottlenecks during discovery and development of biotherapeutics. Computational tools that can predict aggregation prone regions as well as T- and B-cell immune epitopes from protein sequence and structure have become available recently. Here, we describe a potential coupling between aggregation and immunogenicity: T-cell and B-cell immune epitopes in therapeutic proteins may contain aggregation-prone regions. The details of biological mechanisms behind this observation remain to be understood. However, our observation opens up an exciting potential for rational design of de-immunized novel, as well as follow on biotherapeutics with reduced aggregation propensity.

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

    PubMed

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

    2013-09-13

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

  11. Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

    SciTech Connect

    Yang, Marie; Mine, Yoshinori

    2009-01-09

    The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e., A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-{gamma} and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.

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

    PubMed

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

    2014-05-30

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

  13. Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

    PubMed

    Holtappels, Rafaela; Simon, Christian O; Munks, Michael W; Thomas, Doris; Deegen, Petra; Kühnapfel, Birgit; Däubner, Torsten; Emde, Simone F; Podlech, Jürgen; Grzimek, Natascha K A; Oehrlein-Karpi, Silke A; Hill, Ann B; Reddehase, Matthias J

    2008-06-01

    Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-04-01

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

  16. Escape from a Dominant HLA-B*15-Restricted CD8+ T Cell Response against Hepatitis C Virus Requires Compensatory Mutations outside the Epitope

    PubMed Central

    Ruhl, Marianne; Chhatwal, Patrick; Strathmann, Heiko; Kuntzen, Thomas; Bankwitz, Dorothea; Skibbe, Kathrin; Walker, Andreas; Heinemann, Falko M.; Horn, Peter A.; Allen, Todd M.; Hoffmann, Daniel; Pietschmann, Thomas

    2012-01-01

    Antiviral CD8+ T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8+ T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8+ epitopes in NS5B (LLRHHNMVY2450-2458 and SQRQKKVTF2466-2474), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs. PMID:22072759

  17. Murine whole-organ immune cell populations revealed by multi-epitope-ligand cartography.

    PubMed

    Eckhardt, Jenny; Ostalecki, Christian; Kuczera, Katarzyna; Schuler, Gerold; Pommer, Ansgar J; Lechmann, Matthias

    2013-02-01

    Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy-based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR.

  18. Murine Whole-Organ Immune Cell Populations Revealed by Multi-epitope-Ligand Cartography

    PubMed Central

    Eckhardt, Jenny; Ostalecki, Christian; Kuczera, Katarzyna; Schuler, Gerold; Lechmann, Matthias

    2013-01-01

    Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy–based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR. PMID:23160665

  19. Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen.

    PubMed

    Wu, Han-Chung; Jung, Mei-Ying; Chiu, Chien-Yu; Chao, Ting-Ting; Lai, Szu-Chia; Jan, Jia-Tsrong; Shaio, Men-Fang

    2003-10-01

    In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  2. Oral Immunotherapy for Pollen Allergy Using T-Cell Epitope-Containing Egg White Derived from Genetically Manipulated Chickens

    PubMed Central

    Kawabe, Yoshinori; Hayashida, Yuuki; Numata, Kensaku; Harada, Shota; Hayashida, Yoshifumi; Ito, Akira; Kamihira, Masamichi

    2012-01-01

    Peptide immunotherapy using T-cell epitopes is expected to be an effective treatment for allergic diseases such as Japanese cedar (Cryptomeria japonica; Cj) pollinosis. To develop a treatment for pollen allergy by inducing oral tolerance, we generated genetically manipulated (GM) chickens by retroviral gene transduction, to produce a fusion protein of chicken egg white lysozyme and a peptide derived from seven dominant human T-cell epitopes of Japanese cedar pollen allergens (cLys-7crp). The transgene sequence was detected in all chickens transduced with the retroviral vector. Transduction efficiency in blood cells correlated to transgene expression. Western blot analysis revealed that cLys-7crp was expressed in the egg white of GM hens. Mice induced to develop allergic rhinitis by Cj pollinosis were fed with cLys-7crp-containing egg white produced by GM chickens. Total and Cj allergen (Cry j 1)-specific IgE levels were significantly decreased in allergic mice fed with cLys-7crp-containing egg white compared with allergic mice fed with normal egg white. These results suggest that oral administration of T-cell epitope-containing egg white derived from GM chickens is effective for the induction of immune tolerance as an allergy therapy. PMID:23144766

  3. Microfluidic jet injection for delivering macromolecules into cells

    PubMed Central

    Adamo, A.; Roushdy, O.; Dokov, R.; Sharei, A.; Jensen, K.F.

    2013-01-01

    We present a microfluidic based injection system designed to achieve intracellular delivery of macromolecules by directing a picoliter-jet of a solution towards individual cells. After discussing the concept, we present design specification and criteria, elucidate performance and discuss results. The method has the potential to be quantitative and high throughput, overcoming limitations of current intracellular delivery protocols. PMID:23956498

  4. Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

    PubMed Central

    Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

    2014-01-01

    We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

  5. Lymphocyte response to tetanus toxin T-cell epitopes: effects of tetanus vaccination and concurrent malaria prophylaxis.

    PubMed

    Fryauff, D J; Mouzin, E; Church, L W; Ratiwayanto, S; Hadiputranto, H; Sutamihardja, M A; Widjaja, H; Corradin, G; Subianto, B; Hoffman, S L

    1999-01-01

    Synthesized T-cell epitopes of tetanus toxin are universally immunogenic and serve to enhance immune response when they are used as vaccine carriers of B-cell epitopes. The immunogenicity of the P2, P30, and P2P30 T-cell epitopes of tetanus toxin and whole tetanus toxoid (TT) was evaluated by in vitro proliferation assay of lymphocytes from men with no history of tetanus vaccination who were enrolled in a malaria prophylaxis trial. The enhancement of immune response by tetanus vaccination (Td) and possible antagonism by the antimalarial drugs, was measured by pre- and post-Td comparisons within and between immunized prophylaxis groups (primaquine, chloroquine, placebo) and a nonimmunized control group. Constructs demonstrated low immunogenicity relative to TT in all groups. Relative to both control and its own baseline, the immunized primaquine prophylaxis group was distinct in demonstrating significantly increased proliferation against all three subunits and at both high (30 microg ml(-1)) and low (3 microg ml(-1)) concentrations. Immunization elicited significantly increased proliferation responses by placebo and chloroquine prophylaxis groups against only the P2P30 construct. Despite these significant post-Td changes, a low concentration of TT 0.1 microg ml(-1)) stimulated proliferation 7-10 times over that induced by the greatest concentration of the constructs.

  6. Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1

    PubMed Central

    Greineder, Colin F.; Villa, Carlos H.; Hood, Elizabeth D.; Shuvaev, Vladimir V.; Sun, Jing; Chacko, Ann-Marie; Abraham, Valsamma; DeLisser, Horace M.; Muzykantov, Vladimir R.

    2017-01-01

    Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications. PMID:28085903

  7. Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

    PubMed

    Kiseleva, Raisa; Greineder, Colin F; Villa, Carlos H; Hood, Elizabeth D; Shuvaev, Vladimir V; Sun, Jing; Chacko, Ann-Marie; Abraham, Valsamma; DeLisser, Horace M; Muzykantov, Vladimir R

    2017-01-01

    Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

  8. Computational methods for prediction of T-cell epitopes--a framework for modelling, testing, and applications.

    PubMed

    Brusic, Vladimir; Bajic, Vladimir B; Petrovsky, Nikolai

    2004-12-01

    Computational models complement laboratory experimentation for efficient identification of MHC-binding peptides and T-cell epitopes. Methods for prediction of MHC-binding peptides include binding motifs, quantitative matrices, artificial neural networks, hidden Markov models, and molecular modelling. Models derived by these methods have been successfully used for prediction of T-cell epitopes in cancer, autoimmunity, infectious disease, and allergy. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures and performed according to strict standards. This requires careful selection of data for model building, and adequate testing and validation. A range of web-based databases and MHC-binding prediction programs are available. Although some available prediction programs for particular MHC alleles have reasonable accuracy, there is no guarantee that all models produce good quality predictions. In this article, we present and discuss a framework for modelling, testing, and applications of computational methods used in predictions of T-cell epitopes.

  9. Recognition of peptide epitopes of the 16,000 MW antigen of Mycobacterium tuberculosis by murine T cells.

    PubMed Central

    Vordermeier, H M; Harris, D P; Lathigra, R; Roman, E; Moreno, C; Ivanyi, J

    1993-01-01

    The T-cell repertoire to a prominent immunogen of Mycobacterium tuberculosis has been investigated on the assumption that differences in epitope specificity could influence the protective and pathogenic host reactions. Proliferative responses of lymph node and spleen cells to overlapping peptides, spanning the entire sequence of the 16,000 MW protein antigen were analysed in C57BL/10 and B10.BR mice. Following footpad priming and in vitro challenge with homologous peptide, 12 out of the 14 peptides tested were found to be immunogenic. However, only two peptides of residues 31-40 and 71-91 stimulated strong proliferative responses of T cells from mice which had been presensitized with either killed or live M. tuberculosis organisms; another three peptides were only weakly stimulatory. These epitopes have been immunodominant in both H-2b and H-2k mouse strains, indicating the genetically permissive nature of their recognition. Furthermore, both major immunodominant epitopes were found to be species specific for the M. tuberculosis complex and therefore potentially suitable for the early diagnosis of tuberculous infection. PMID:7503946

  10. How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells?

    PubMed Central

    Petre, Benjamin; Kamoun, Sophien

    2014-01-01

    Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens. PMID:24586116

  11. Software tools for simultaneous data visualization and T cell epitopes and disorder prediction in proteins.

    PubMed

    Jandrlić, Davorka R; Lazić, Goran M; Mitić, Nenad S; Pavlović, Mirjana D

    2016-04-01

    We have developed EpDis and MassPred, extendable open source software tools that support bioinformatic research and enable parallel use of different methods for the prediction of T cell epitopes, disorder and disordered binding regions and hydropathy calculation. These tools offer a semi-automated installation of chosen sets of external predictors and an interface allowing for easy application of the prediction methods, which can be applied either to individual proteins or to datasets of a large number of proteins. In addition to access to prediction methods, the tools also provide visualization of the obtained results, calculation of consensus from results of different methods, as well as import of experimental data and their comparison with results obtained with different predictors. The tools also offer a graphical user interface and the possibility to store data and the results obtained using all of the integrated methods in the relational database or flat file for further analysis. The MassPred part enables a massive parallel application of all integrated predictors to the set of proteins. Both tools can be downloaded from http://bioinfo.matf.bg.ac.rs/home/downloads.wafl?cat=Software. Appendix A includes the technical description of the created tools and a list of supported predictors.

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

    PubMed Central

    Hossain, Md. Saddam; Chowdhury, Parveen Afroz; Wakayama, Mamoru

    2017-01-01

    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. PMID:28401156

  13. The Use of Reverse Vaccinology and Molecular Modeling Associated with Cell Proliferation Stimulation Approach to Select Promiscuous Epitopes from Schistosoma mansoni.

    PubMed

    Oliveira, Flávio M; Coelho, Ivan E V; Lopes, Marcelo D; Taranto, Alex G; Junior, Moacyr C; Santos, Luciana L D; Villar, José A P F; Fonseca, Cristina T; Lopes, Débora D O

    2016-07-01

    Schistosomiasis remains an important parasitic disease that affects millions of individuals worldwide. Despite the availability of chemotherapy, the occurrence of constant reinfection demonstrates the need for additional forms of intervention and the development of a vaccine represents a relevant strategy to control this disease. With the advent of genomics and bioinformatics, new strategies to search for vaccine targets have been proposed, as the reverse vaccinology. In this work, computational analyses of Schistosoma mansoni membrane proteins were performed to predict epitopes with high affinity for different human leukocyte antigen (HLA)-DRB1. Ten epitopes were selected and along with murine major histocompatibility complex (MHC) class II molecule had their three-dimensional structures optimized. Epitope interactions were evaluated against murine MHC class II molecule through molecular docking, electrostatic potential, and molecular volume. The epitope Sm141290 and Sm050890 stood out in most of the molecular modeling analyses. Cellular proliferation assay was performed to evaluate the ability of these epitopes to bind to murine MHC II molecules and stimulate CD4+ T cells showing that the same epitopes were able to significantly stimulate cell proliferation. This work showed an important strategy of peptide selection for epitope-based vaccine design, achieved by in silico analyses that can precede in vivo and in vitro experiments, avoiding excessive experimentation.

  14. CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope.

    PubMed

    Mak, Anthony B; Blakely, Kim M; Williams, Rashida A; Penttilä, Pier-Andrée; Shukalyuk, Andrey I; Osman, Khan T; Kasimer, Dahlia; Ketela, Troy; Moffat, Jason

    2011-11-25

    The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

  15. Vaccine Targeting of Subdominant CD8+ T Cell Epitopes Increases the Breadth of the T Cell Response upon Viral Challenge, but May Impair Immediate Virus Control.

    PubMed

    Steffensen, Maria A; Pedersen, Louise H; Jahn, Marie L; Nielsen, Karen N; Christensen, Jan P; Thomsen, Allan R

    2016-03-15

    As a result of the difficulties in making efficient vaccines against genetically unstable viruses such as HIV, it has been suggested that future vaccines should preferentially target subdominant epitopes, the idea being that this should allow a greater breadth of the induced T cell response and, hence, a greater efficiency in controlling escape variants. However, to our knowledge the evidence supporting this concept is limited at best. To improve upon this, we used the murine lymphocytic choriomeningitis virus model and adenoviral vectors to compare a vaccine expressing unmodified Ag to a vaccine expressing the same Ag without its immunodominant epitope. We found that removal of the dominant epitope allowed the induction of CD8(+) T cell responses targeting at least two otherwise subdominant epitopes. Importantly, the overall magnitude of the induced T cell responses was similar, allowing us to directly compare the efficiency of these vaccines. Doing this, we observed that mice vaccinated with the vaccine expressing unmodified Ag more efficiently controlled an acute viral challenge. In the course of a more chronic viral infection, mice vaccinated using the vaccine targeting subdominant epitopes caught up with the conventionally vaccinated mice, and analysis of the breadth of the CD8(+) T cell response revealed that this was notably greater in the former mice. However, under the conditions of our studies, we never saw any functional advantage of this. This may represent a limitation of our model, but clearly our findings underscore the importance of carefully weighing the pros and cons of changes in epitope targeting before any implementation. Copyright © 2016 by The American Association of Immunologists, Inc.

  16. Pep-3D-Search: a method for B-cell epitope prediction based on mimotope analysis

    PubMed Central

    Huang, Yan Xin; Bao, Yong Li; Guo, Shu Yan; Wang, Yan; Zhou, Chun Guang; Li, Yu Xin

    2008-01-01

    Background The prediction of conformational B-cell epitopes is one of the most important goals in immunoinformatics. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues of interaction between an antigen and an antibody. Consequently, this area of research has received considerable attention from immunologists, structural biologists and computational biologists. Phage-displayed random peptide libraries are powerful tools used to obtain mimotopes that are selected by binding to a given monoclonal antibody (mAb) in a similar way to the native epitope. These mimotopes can be considered as functional epitope mimics. Mimotope analysis based methods can predict not only linear but also conformational epitopes and this has been the focus of much research in recent years. Though some algorithms based on mimotope analysis have been proposed, the precise localization of the interaction site mimicked by the mimotopes is still a challenging task. Results In this study, we propose a method for B-cell epitope prediction based on mimotope analysis called Pep-3D-Search. Given the 3D structure of an antigen and a set of mimotopes (or a motif sequence derived from the set of mimotopes), Pep-3D-Search can be used in two modes: mimotope or motif. To evaluate the performance of Pep-3D-Search to predict epitopes from a set of mimotopes, 10 epitopes defined by crystallography were compared with the predicted results from a Pep-3D-Search: the average Matthews correlation oefficient (MCC), sensitivity and precision were 0.1758, 0.3642 and 0.6948. Compared with other available prediction algorithms, Pep-3D-Search showed comparable MCC, specificity and precision, and could provide novel, rational results. To verify the capability of Pep-3D-Search to align a motif sequence to a 3D structure for predicting epitopes, 6 test cases were used. The predictive performance of Pep-3D-Search was demonstrated to be superior to that of other

  17. The Alpha Stem Cell Clinic: A Model for Evaluating and Delivering Stem Cell-Based Therapies

    PubMed Central

    DeWitt, Natalie D.; Feigal, Ellen G.

    2012-01-01

    Summary Cellular therapies require the careful preparation, expansion, characterization, and delivery of cells in a clinical environment. There are major challenges associated with the delivery of cell therapies and high costs that will limit the companies available to fully evaluate their merit in clinical trials, and will handicap their application at the present financial environment. Cells will be manufactured in good manufacturing practice or near-equivalent facilities with prerequisite safety practices in place, and cell delivery systems will be specialized and require well-trained medical and nursing staff, technicians or nurses trained to handle cells once delivered, patient counselors, as well as statisticians and database managers who will oversee the monitoring of patients in relatively long-term follow-up studies. The model proposed for Alpha Stem Cell Clinics will initially use the capacities and infrastructure that exist in the most advanced tertiary medical clinics for delivery of established bone marrow stem cell therapies. As the research evolves, they will incorporate improved procedures and cell preparations. This model enables commercialization of medical devices, reagents, and other products required for cell therapies. A carefully constructed cell therapy clinical infrastructure with the requisite scientific, technical, and medical expertise and operational efficiencies will have the capabilities to address three fundamental and critical functions: 1) fostering clinical trials; 2) evaluating and establishing safe and effective therapies, and 3) developing and maintaining the delivery of therapies approved by the Food and Drug Administration, or other regulatory agencies. PMID:23197634

  18. Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies.

    PubMed

    LaVecchio, J A; Dunne, A D; Edge, A S

    1995-10-27

    Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.

  19. Identification of the Neutralizing Epitopes of Merkel Cell Polyomavirus Major Capsid Protein within the BC and EF Surface Loops

    PubMed Central

    Fleury, Maxime J. J.; Nicol, Jérôme T. J.; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  20. Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

    PubMed

    Focke, M; Mahler, V; Ball, T; Sperr, W R; Majlesi, Y; Valent, P; Kraft, D; Valenta, R

    2001-09-01

    Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.

  1. Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses.

    PubMed

    Kutubuddin, M; Kolaskar, A S; Galande, S; Gore, M M; Ghosh, S N; Banerjee, K

    1991-01-01

    Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.

  2. The challenges and opportunities for the development of a T-cell epitope-based herpes simplex vaccine.

    PubMed

    Kuo, Tiffany; Wang, Christine; Badakhshan, Tina; Chilukuri, Sravya; BenMohamed, Lbachir

    2014-11-28

    Herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) infections have been prevalent since the ancient Greek times. To this day, they still affect a staggering number of over a billion individuals worldwide. HSV-1 infections are predominant than HSV-2 infections and cause potentially blinding ocular herpes, oro-facial herpes and encephalitis. HSV-2 infections cause painful genital herpes, encephalitis, and death in newborns. While prophylactic and therapeutic HSV vaccines remain urgently needed for centuries, their development has been difficult. During the most recent National Institute of Health (NIH) workshop titled "Next Generation Herpes Simplex Virus Vaccines: The Challenges and Opportunities", basic researchers, funding agencies, and pharmaceutical representatives gathered: (i) to assess the status of herpes vaccine research; and (ii) to identify the gaps and propose alternative approaches in developing a safe and efficient herpes vaccine. One "common denominator" among previously failed clinical herpes vaccine trials is that they either used a whole virus or a whole viral protein, which contain both "pathogenic symptomatic" and "protective asymptomatic" antigens and epitopes. In this report, we continue to advocate developing "asymptomatic" epitope-based sub-unit vaccine strategies that selectively incorporate "protective asymptomatic" epitopes which: (i) are exclusively recognized by effector memory CD4(+) and CD8(+) T cells (TEM cells) from "naturally" protected seropositive asymptomatic individuals; and (ii) protect human leukocyte antigen (HLA) transgenic animal models of ocular and genital herpes. We review the role of animal models in herpes vaccine development and discuss their current status, challenges, and prospects.

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

    PubMed Central

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

    2013-01-01

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

  4. A powerful combination: the use of positional scanning libraries and biometrical analysis to identify cross-reactive T cell epitopes.

    PubMed

    Nino-Vasquez, J Javier; Allicotti, Gina; Borras, Eva; Wilson, Darcy B; Valmori, Danila; Simon, Richard; Martin, Roland; Pinilla, Clemencia

    2004-02-01

    Studies on the elucidation of the specificity of the T cell receptor (TCR) at the antigen and peptide level have contributed to the current understanding of T cell cross-reactivity. Historically, most studies of T cell specificity and degeneracy have relied on the determination of the effects on T cell recognition of amino acid changes at individual positions or MHC binding residues, and thus they have been limited to a small set of possible ligands. Synthetic combinatorial libraries (SCLs), and in particular positional scanning synthetic combinatorial libraries (PS-SCLs) represent collections of millions to trillions of peptides which allow the unbiased elucidation of T cell ligands that stimulate clones of both known and unknown specificity. PS-SCLs have been used successfully to study T cell recognition and to identify and optimize T cell clone (TCC) epitopes in infectious diseases, autoimmune disorders and tumor antigens. PS-SCL-based biometrical analysis represents a further refinement in the analysis of the data derived from the screening of a library with a TCC. It combines this data with information derived from protein sequence databases to identify natural peptide ligands. PS-SCL-based biometrical analysis provides a method for the determination of new microbial antigen and autoantigen sequences based solely on functional data rather than sequence homology or motifs, making the method ideally suited for the prediction and identification of both native and cross-reactive epitopes by virtue of its ability to integrate the examination of trillions of peptides in a systematic manner with all of the protein sequences in a given database. We review here the application of PS-SCLs and biometrical analysis to identify cross-reactive T cell epitopes, as well as the current efforts to refine this strategy.

  5. Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes.

    PubMed

    Gunnarsson, Anders Patrik; Christensen, Rikke; Praetorius, Jeppe; Jensen, Uffe Birk

    2017-01-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive basal cells express higher levels of Itgβ1 the colony-forming efficiency is slightly lower than CSPG4-negative basal cells. Sorting the directly isolated keratinocytes based on Itgβ1 did not reveal differences in colony-forming efficiency between keratinocytes expressing high or low levels of Itgβ1. However, after the first passage Itgβ1 could be used to predict colony-forming efficiency whether the culture was established from CSPG4-positive or CSPG4-negative basal cell keratinocytes. Although we were unable to detect differences in the colony-forming assay, global gene expression profiling showed that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges the way we assess for stemness within the human epidermal basal layer.

  6. An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

    PubMed Central

    Enomoto-Okawa, Yurie; Maeda, Yuka; Harashima, Nozomi; Sugawara, Yumika; Katagiri, Fumihiko; Hozumi, Kentaro; Hui, Kam Man; Nomizu, Motoyoshi; Ito, Yuji; Kikkawa, Yamato

    2017-01-01

    The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg247 is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis. PMID:28060819

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

    SciTech Connect

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

    2006-05-26

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

  8. Susceptibility to chronic hepatitis C virus infection is influenced by sequence differences in immunodominant CD8+ T cell epitopes.

    PubMed

    Ziegler, Susanne; Ruhl, Marianne; Tenckhoff, Hannelore; Wiese, Manfred; Heinemann, Falko M; Horn, Peter A; Spengler, Ulrich; Neumann-Haefelin, Christoph; Nattermann, Jacob; Timm, Jörg

    2013-01-01

    The antiviral immune response against HCV by CD8+ T cells plays a central role in viral containment. In a large HCV genotype 1b outbreak in Ireland, HLA-B(∗)08 was identified as a risk allele for chronic infection and HLA-A(∗)03 and HLA-B(∗)27 were associated with higher clearance rates. Here we took advantage of a similar large common source HCV genotype 1b outbreak (East-German cohort) to determine the role of HLA class I alleles and the sequence of the infection source, in immunodominant CD8+ T cell epitopes for disease outcome. HLA-type and IL28B genotype were determined in 216 patients with chronic and 95 with spontaneously resolved HCV infection. The viral sequence in immunodominant epitopes was determined in the infection source and in patients with chronic infection. In contrast to the Irish cohort, HLA-B(∗)08, HLA-A(∗)03 and HLA-B(∗)27 were neutral for disease outcome even when the cohort was stratified for the IL28B genotype. Sequence analysis of the immunodominant epitopes revealed that pre-existing substitutions in the infection source of both cohorts influenced the impact of the corresponding HLA-allele. The immunodominant epitopes presented by the "protective" alleles HLA-A(∗)03 and -B(∗)27 in the Irish cohort contained substitutions in the source virus of the East-German outbreak. Importantly, the pre-existing substitutions altered subsequent selection pressure and viral evolution in the East-German cohort. This study highlights that subtle sequence differences in the infection source may have profound effects on the ability to clear HCV infection in the presence of particular HLA class I alleles. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  9. Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts

    PubMed Central

    Archila, Luis Diego; Chow, I-Ting; McGinty, John W.; Renand, Amedee; Jeong, David; Robinson, David; Farrington, Mary L.; Kwok, William.W.

    2017-01-01

    Background Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. Objectives In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. Methods CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. Results CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH2 or TH2/TH17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Conclusions Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH2 type response at an epitope specific level. Clinical relevance Lack of cross-reactivity between walnut and cashew suggest that cashew peptide immunotherapy approach may not be most effective for walnut. PMID:27129138

  10. Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

    PubMed

    Archila, L D; Chow, I-T; McGinty, J W; Renand, A; Jeong, D; Robinson, D; Farrington, M L; Kwok, W W

    2016-06-01

    Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut. © 2016 John Wiley & Sons Ltd.

  11. The immunological footprint of Mycobacterium tuberculosis T-cell epitope recognition.

    PubMed

    Axelsson-Robertson, Rebecca; Magalhaes, Isabelle; Parida, Shreemanta K; Zumla, Alimuddin; Maeurer, Markus

    2012-05-15

    Aerosols containing Mycobacterium tuberculosis (MTB) generated from the cough of patients with active pulmonary tuberculosis are the source of MTB infection. About 70% of individuals exposed to infected aerosols do not get infected, depending on the intensity and duration of MTB exposure. Only 40% of the rest of the individuals (about 10% of those originally exposed) develop primary tuberculosis, whereas the remaining 60% contain the infection with generation of a robust immune response leading to latent tuberculosis, which is regarded as a spectrum rather than a single entity. The mechanisms involved in this natural protection are not yet well understood. There is an increasing need to integrate all disparate observations into a coherent systems biology approach for a comprehensive understanding: we need to decipher the nature of success and failure in MTB infection in humans. New advances in cellular immunology will aid in achieving that goal. We review here the nature of MTB peptide generation, antigen presentation, and detection of major histocompatibility complex class I and II-presented T-cell epitopes. Cross-sectional thinking from lessons learned in the context of the major efforts to develop vaccines will help to dissect biologically relevant mechanisms that need to be translated into the clinical context of MTB infection with the aim to (1) better understand clinically relevant T-cell responses in individuals protected from tuberculosis disease and develop markers of immune protection and vaccine take, (2) characterize the nature of the immune response in individuals who are not able to contain MTB infection, and ultimately (3) characterize markers to gauge response to therapy.

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

    PubMed

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    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.

  13. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31

    PubMed Central

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; Ryu, Chun Jeih

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments. PMID:27907150

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

    PubMed Central

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

    2016-01-01

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

  15. Type II and III Receptors for Immunoglobulin G (IgG) Control the Presentation of Different T Cell Epitopes from Single IgG-complexed Antigens

    PubMed Central

    Amigorena, Sebastian; Lankar, Danielle; Briken, Volker; Gapin, Laurent; Viguier, Mireille; Bonnerot, Christian

    1998-01-01

    T cell receptors on CD4+ lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcγRIIb2 and FcγRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcγRIIb2 or FcγRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcγRIIb2 and FcγRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcγRIII, but not through FcγRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcγRIIb2 by addition of the FcγRIII-associated γ chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the γ chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens. PMID:9463401

  16. Identification of B-cell epitopes on virus-like particles of cutaneous alpha-human papillomaviruses.

    PubMed

    Senger, Tilo; Becker, Maria R; Schädlich, Lysann; Waterboer, Tim; Gissmann, Lutz

    2009-12-01

    Human papillomavirus (PV) (HPV) types 2, 27, and 57 are closely related and, hence, represent a promising model system to study the correlation of phylogenetic relationship and immunological distinctiveness of PVs. These HPV types cause a large fraction of cutaneous warts occurring in immunocompromised patients. Therefore, they constitute a target for the development of virus-like particle (VLP)-based vaccines. However, the immunogenic structure of HPV type 2, 27, and 57 capsids has not been studied yet. Here we provide, for the first time, a characterization of the B-cell epitopes on VLPs of cutaneous alpha-HPVs using a panel of 94 monoclonal antibodies (MAbs) generated upon immunization with capsids from HPV types 2, 27, and 57. The MAbs generated were characterized regarding their reactivities with glutathione S-transferase-L1 fusion proteins from 18 different PV types, the nature of their recognized epitopes, their isotypes, and their ability to neutralize HPV type 2, 27, 57, or 16. In total, 33 of the 94 MAbs (35%) showed type-specific reactivity. All type-specific MAbs recognize linear epitopes, most of which map to the hypervariable surface loop regions of the L1 amino acid sequence. Four of the generated MAbs neutralized pseudovirions of the inoculated HPV type efficiently. All four MAbs recognized epitopes within the BC loop, which is required and sufficient for their neutralizing activity. Our data highlight the immunological distinctiveness of individual HPV types, even in comparison to their closest relatives, and they provide a basis for the development of VLP-based vaccines against cutaneous alpha-HPVs.

  17. Mapping myasthenia gravis–associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice

    PubMed Central

    Yang, Huan; Goluszko, Elzbieta; David, Chella; Okita, David K.; Conti-Fine, Bianca; Chan, Teh-sheng; Poussin, Mathilde A.; Christadoss, Premkumar

    2002-01-01

    Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8×DQ6 and DQ8×DR3 F1 transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (α320-337, α304-322, and α419-437), of which the response to α320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR– and α320-337 peptide–specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG2b, and IgG2c Ab’s and reduced IL-2 and IFN-γ secretion by H-AChR– and peptide α320-337–specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence α320-337 as a dominant trait. PMID:11956249

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

    PubMed

    Machkovech, Heather M; Bedford, Trevor; Suchard, Marc A; Bloom, Jesse D

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-16

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2010-02-20

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

  3. Dendritic Cells Transfected with a DNA Construct Encoding Tumour-associated Antigen Epitopes Induce a Cytotoxic Immune Response Against Autologous Tumour Cells in a Culture of Mononuclear Cells from Colorectal Cancer Patients.

    PubMed

    Kulikova, E V; Kurilin, V V; Shevchenko, J A; Obleukhova, I A; Khrapov, E A; Boyarskikh, U A; Filipenko, M L; Shorokhov, R V; Yakushenko, V K; Sokolov, A V; Sennikov, S V

    2015-08-01

    Significant effort has been devoted to developing effective cancer vaccines based on dendritic cells (DCs) loaded with various tumour antigens, including DNA constructs that carry sequences of tumour-associated antigens (TAAs). Such vaccines efficiently and selectively activate the T cell immune response. In this study, we describe a method to induce an antitumour immune response in mononuclear cell (MNC) cultures from colorectal cancer patients using DNA-transfected DCs encoding TAA epitopes of carcinoembryonic antigen, epithelial cell adhesion molecule and mucin 4. DCs were obtained from peripheral blood monocytes of colorectal cancer patients. Magnetic-assisted transfection was used to deliver the genetic constructs to DCs. To assess the potency of the immune response, the antitumour cytotoxic response was assessed by lymphocyte intracellular perforin and the MNC cytotoxic activity against autologous tumour cells. We showed that polyepitope DNA-transfected DCs enhanced MNC antitumour activity, increasing tumour cell death and the percentage of perforin-positive lymphocytes. In addition, DNA-transfected DCs elicited a cytotoxic response that was as efficient as that of tumour lysate-loaded DCs. Taken together, the data suggest that it is feasible to induce an antitumour immune response in colorectal MNCs using transfected DCs. Thus, the DNA construct reported in this study may potentially be used in therapeutic and prophylactic DC-based vaccines.

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

    PubMed

    Blanco, Esther; Guerra, Beatriz; de la Torre, Beatriz G; Defaus, Sira; Dekker, Aldo; Andreu, David; Sobrino, Francisco

    2016-05-01

    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)] of FMDV [B4T(thi)] elicits potent B- and T-cell specific responses and confers solid protection in pigs to type C FMDV challenge. Herein we show that downsized versions of this peptide bearing two copies of a B-cell epitope from a type O isolate and using thioether [B2T(thi)] or maleimide [B2T(mal)] conjugation chemistries for their synthesis elicited in swine similar or higher B and T-cell specific responses than tetravalent B4T(thi). Moreover, while partial protection was observed in animals immunized with B4T(thi) (60%) and B2T(thi) (80%), B2T(mal) conferred full (100%) protection against FMDV challenge, associated to high levels of circulating IgG2 and mucosal IgGA, and entirely prevented virus shedding. Interestingly, B2T(mal) is also the most advantageous option in terms of synthetic practicality. Taken together, the results reported here point out to B2T(mal) as a highly valuable, cost-effective FMDV candidate vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    1993-01-01

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

  6. Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis

    PubMed Central

    Baggi, Fulvio; Andreetta, Francesca; Caspani, Elisabetta; Milani, Monica; Longhi, Renato; Mantegazza, Renato; Cornelio, Ferdinando; Antozzi, Carlo

    1999-01-01

    The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell–dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell–dependent disease. We report that oral administration of the T-cell epitope α146-162 of the Torpedo californica acetylcholine receptor (TAChR) α-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab’s to mouse AChR and reduced AChR loss in muscle. The effect of Tα146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Tα146-162 was mediated by reduced production of IFN-γ, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-β–secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease. PMID:10545527

  7. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    PubMed

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties.

    PubMed

    Verma, Bhavna; Hawkins, Oriana E; Neethling, Francisca A; Caseltine, Shannon L; Largo, Sherly R; Hildebrand, William H; Weidanz, Jon A

    2010-04-01

    The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.

  12. Differential fates of biomolecules delivered to target cells via extracellular vesicles

    PubMed Central

    Kanada, Masamitsu; Bachmann, Michael H.; Hardy, Jonathan W.; Frimannson, Daniel Omar; Bronsart, Laura; Wang, Andrew; Sylvester, Matthew D.; Schmidt, Tobi L.; Kaspar, Roger L.; Matin, A. C.; Contag, Christopher H.

    2015-01-01

    Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell–cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer. PMID:25713383

  13. Japanese encephalitis protein vaccine candidates expressing neutralizing epitope and M.T hsp70 induce virus-specific memory B cells and long-lasting antibodies in swine.

    PubMed

    Fei-fei, Ge; Jian, Wang; Feng, Xu; Li-ping, Sheng; Quan-yun, Sun; Jin-ping, Zhou; Pu-yan, Chen; Pei-hong, Liu

    2008-10-16

    Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE protein vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids are based on a prokaryotic vector pET-32a(+). One plasmid, designated pET-32a(+)-epitope, encode a cassette consisting of a neutralizing epitope on envelope (E) protein of JE virus, whereas the other plasmid, designated pET-32a(+)-epitope-hsp70, express the fusion protein of the epitope and M.T hsp70. Some differences were detected in the immunogenicity of these two proteins in swine. Swine immunized twice with 2000pmol of the neutralizing epitope or the fusion protein developed neutralizing antibody titers of respectively, 154 and 300, and anti-neutralizing epitope antibody titers of 10(4.25) and 10(6.0) by 3 weeks after the second immunization. In addition, swine immunized with the neutralizing epitope emulsified with adjuvant S206 or with imported mineral oil and Tween-80 induced neutralizing antibody titers of 196 and 244, and anti-neutralizing epitope antibody titers of 10(5.25) or 10(5.6) at the same time point. However, swine administered two doses of a commercial JE vaccine (attenuated virus preparation; JEV SA14-14-2 strain) developed less favorable antibody responses with neutralizing antibody titer 40 and anti-neutralizing epitope antibody titers 10(3.7). The anamnestic response was followed by monitoring titers 1 week after boosting with a viral antigen; swine immunized twice with the fusion protein showed a 177-fold increase in anti-neutralizing epitope titer, indicating a strong recall of the antibody response. The animals maintained detectable levels of anti-neutralizing epitope antibody for at least 105 days after two immunizations, indicating that these four protein antigens are able to stimulate virus-specific memory B cells and long-lasting antibodies at higher levels than is achieved using a current commercial attenuated JEV vaccine

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed

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

    2008-07-23

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

  16. Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1.

    PubMed

    Tokarev, Andrey; Stoneham, Charlotte; Lewinski, Mary K; Mukim, Amey; Deshmukh, Savitha; Vollbrecht, Thomas; Spina, Celsa A; Guatelli, John

    2015-12-16

    HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. Pathogenic viruses encode gene products that enable

  17. A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway

    PubMed Central

    Helling, Bianca; König, Martin; Dälken, Benjamin; Engling, Andre; Krömer, Wolfgang; Heim, Katharina; Wallmeier, Holger; Haas, Jürgen; Wildemann, Brigitte; Fritz, Brigitte; Jonuleit, Helmut; Kubach, Jan; Dingermann, Theodor; Radeke, Heinfried H; Osterroth, Frank; Uherek, Christoph; Czeloth, Niklas; Schüttrumpf, Jörg

    2015-01-01

    CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing. PMID:25512343

  18. In silico prediction of the T-cell and IgE-binding epitopes of Per a 6 and Bla g 6 allergens in cockroaches.

    PubMed

    Chen, Hao; Yang, Hai-Wei; Wei, Ji-Fu; Tao, Ai-Lin

    2014-10-01

    Per a 6 and Bla g 6 are cockroach allergens found in Periplaneta americana and Blattella germanica, respectively. The objective of the present study was to predict the B‑ and T‑cell epitopes of the Per a 6 and Bla g 6 allergens. Three immunoinformatics tools, the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides 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 the two allergens. As a result, seven peptides were predicted in the Per a 6 allergen and seven peptides were predicted in the Bla g 6 allergen in the B‑cell epitope predictions. In the T‑cell prediction, the Per a 6 allergen was predicted to have nine strongly binding nonamer core epitope sequences (IC50<50 nm) and 28 weakly binding sequences (50 nm

  19. FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes.

    PubMed

    Pilarski, L M; Turley, E A; Shaw, A R; Gallatin, W M; Laderoute, M P; Gillitzer, R; Beckman, I G; Zola, H

    1991-07-01

    In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1

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

    PubMed Central

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

    2014-01-01

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

  1. Combining computer algorithms with experimental approaches permits the rapid and accurate identification of T cell epitopes from defined antigens.

    PubMed

    Schirle, M; Weinschenk, T; Stevanović, S

    2001-11-01

    The identification of T cell epitopes from immunologically relevant antigens remains a critical step in the development of vaccines and methods for monitoring of T cell responses. This review presents an overview of strategies that employ computer algorithms for the selection of candidate peptides from defined proteins and subsequent verification of their in vivo relevance by experimental approaches. Several computer algorithms are currently being used for epitope prediction of various major histocompatibility complex (MHC) class I and II molecules, based either on the analysis of natural MHC ligands or on the binding properties of synthetic peptides. Moreover, the analysis of proteasomal digests of peptides and whole proteins has led to the development of algorithms for the prediction of proteasomal cleavages. In order to verify the generation of the predicted peptides during antigen processing in vivo as well as their immunogenic potential, several experimental approaches have been pursued in the recent past. Mass spectrometry-based bioanalytical approaches have been used specifically to detect predicted peptides among isolated natural ligands. Other strategies employ various methods for the stimulation of primary T cell responses against the predicted peptides and subsequent testing of the recognition pattern towards target cells that express the antigen.

  2. The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes

    PubMed Central

    Mannering, S I; Pang, S H; Williamson, N A; Naselli, G; Reynolds, E C; O'Brien-Simpson, N M; Purcell, A W; Harrison, L C

    2009-01-01

    Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing β cells. While the role of CD4+ T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4+ T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4+ T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6–A7). Here we describe the analysis of a CD4+ T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4+ T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4+ T cell responses in human T1D. PMID:19292763

  3. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function 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) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

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

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    2006-03-01

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

  6. CD4+ T-cell activation for immunotherapy of malignancies using Ii-Key/MHC class II epitope hybrid vaccines.

    PubMed

    Xu, Minzhen; Kallinteris, Nikoletta L; von Hofe, Eric

    2012-04-16

    Active immunotherapy is becoming a reality in the treatment of malignancies. Peptide-based vaccines represent a simple, safe, and economic basis for cancer immunotherapeutics development. However, therapeutic efficacy has been disappointing. Some of the reasons for this, such as selection of patients with advanced disease and ignorance of the delayed activity of many immunotherapeutic vaccines, have hampered the entire field of cancer immunotherapy over the last decade. Another reason for this may be that most peptide regimens historically have focused on activation of CD8+ cytotoxic T lymphocytes, having little or only indirect CD4+ T helper (Th) cell activation. We review here evidence for the importance of specific CD4+ Th activation in cancer immunotherapy and the use of Ii-Key technology to accomplish this. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii protein), facilitates the direct charging of peptide epitopes onto MHC class II molecules. Directly linking Ii-Key to MHC class II peptide epitopes greatly enhances their potency in activating CD4+ T-cells. The Ii-Key hybrid AE37, generated by linking LRMK to the known HER2 MHC class II epitope HER2 (aa 776-790), has been shown to generate robust, long lasting HER2-specific immune responses both in patients with breast and prostate cancer. Interim data from a phase II study of AE37 in breast cancer patients suggest a possible improvement in clinical outcome. The Ii-Key hybrid technology is compared to other methods for enhancing the potency of peptide immunotherapy for cancer. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2015-02-18

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

  8. Identification of Continuous Human B-Cell Epitopes in the VP35, VP40, Nucleoprotein and Glycoprotein of Ebola Virus

    PubMed Central

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M.

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7–12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods. PMID:24914933

  9. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

    PubMed

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

  10. T cell epitope-specific defects in the immune response to cat allergen in patients with atopic dermatitis.

    PubMed

    Carneiro, Raquel; Reefer, Amanda; Wilson, Barbara; Hammer, Juergen; Platts-Mills, Thomas; Custis, Natalie; Woodfolk, Judith

    2004-04-01

    Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

  11. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    PubMed Central

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

  12. A rice-based edible vaccine expressing multiple T-cell epitopes to induce oral tolerance and inhibit allergy.

    PubMed

    Takaiwa, Fumio

    2007-02-01

    Plant pollens are the most common cause of seasonal allergic disease. The number of patients undergoing treatment for allergies to the pollen of Japanese cedar (major antigens, Cry j 1 and Cry j 2) has increased steadily each year. A rice seed-based edible vaccine has been shown to be effective for treating Japanese cedar pollinosis. Rice seeds containing the major T-cell epitopes derived from cedar pollen allergens were orally administrated to mice before systemic challenge with total pollen protein. Mucosal immune tolerance leading to a reduction of allergen-specific IgE, T-cell proliferative reactions, and histamine were induced, resulting in suppression of allergy-specific symptoms such as sneezing. Oral seed-based peptide immunotherapy offers a safe, simple, and cost-effective alternative to conventional allergen-specific immunotherapy using crude allergen extracts for treating allergic disease. A human version of rice seed-based edible vaccine containing seven T-cell epitopes from the Cry j 1 and Cry j 2 allergens was recently developed and is undergoing safety assessments.

  13. A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes.

    PubMed

    Xiang, Sue D; Gao, Qian; Wilson, Kirsty L; Heyerick, Arne; Plebanski, Magdalena

    2015-10-29

    Sperm protein antigen 17 (Sp17), expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17) sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional "mix-in" pro-inflammatory adjuvant CpG, both mapping to amino acids (aa) 111-142. However, delivery of hSp17111-142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111-142, from an immuno-dominant region 134-142 aa for CpG, to region 121-138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses.

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

    PubMed Central

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

    2015-01-01

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

  15. Strategies to Query and Display Allergy-Derived Epitope Data from the Immune Epitope Database

    PubMed Central

    Vaughan, Kerrie; Peters, Bjoern; Larche, Mark; Pomes, Anna; Broide, David; Sette, Alessandro

    2013-01-01

    The recognition of specific epitopes on allergens by antibodies and T cells is a key element in allergic processes. Analysis of epitope data may be of interest for basic immunopathology or for potential application in diagnostics or immunotherapy. The Immune Epitope Database (IEDB) is a freely available repository of epitope data from infectious disease agents, as well as epitopes defined for allergy, autoimmunity, and transplantation. The IEDB curates the experiments associated with each epitope and thus provides a variety of different ways to search the data. This review aims to demonstrate the utility of the IEDB and its query strategies, including searching by epitope structure (peptidic/nonpeptidic), by assay methodology, by host, by the allergen itself, or by the organism from which the allergen was derived. Links to tools for visualization of 3-D structures, epitope prediction, and analyses of B and T cell reactivity by host response frequency score are also highlighted. PMID:23172234

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

    PubMed Central

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

    2016-01-01

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

  17. HLA Class I-T Cell Epitopes from trans-Sialidase Proteins Reveal Functionally Distinct Subsets of CD8+ T Cells in Chronic Chagas Disease

    PubMed Central

    Alvarez, María G.; Postan, Miriam; Weatherly, D. Brent; Albareda, María C.; Sidney, John; Sette, Alessandro; Olivera, Carina; Armenti, Alejandro H.

    2008-01-01

    Background Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8+ T cell responses in HLA-A0201+ individuals chronically infected by T. cruzi. Methods and Findings Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8+ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8+ T cell responses to promiscuous epitopes revealed that the CD8+ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection. Conclusions This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease. PMID:18846233

  18. T-cell epitopes in type 1 diabetes autoantigen tyrosine phosphatase IA-2: potential for mimicry with rotavirus and other environmental agents.

    PubMed Central

    Honeyman, M. C.; Stone, N. L.; Harrison, L. C.

    1998-01-01

    The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD. PMID:9606176

  19. CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

    PubMed Central

    Skowera, Ania; Ellis, Richard J.; Varela-Calviño, Ruben; Arif, Sefina; Huang, Guo Cai; Van-Krinks, Cassie; Zaremba, Anna; Rackham, Chloe; Allen, Jennifer S.; Tree, Timothy I.M.; Zhao, Min; Dayan, Colin M.; Sewell, Andrew K.; Unger, Wendy; Drijfhout, Jan W.; Ossendorp, Ferry; Roep, Bart O.; Peakman, Mark

    2008-01-01

    The final pathway of β cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill β cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide–specific CD8+ T cells killed human β cells in vitro. Critically, at high glucose concentration, β cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human β cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing β cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining β cells. PMID:18802479

  20. Use of multiple peptides containing T cell epitopes is a feasible approach for peptide-based immunotherapy in Can f 1 allergy

    PubMed Central

    Immonen, Anu K; Taivainen, Antti H; Närvänen, Ale T O; Kinnunen, Tuure T; Saarelainen, Soili A; Rytkönen-Nissinen, Marja A; Virtanen, Tuomas I

    2007-01-01

    We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33–48 and p107–122 favoured the production of interferon-γ and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107–122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy. PMID:17233739

  1. CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

    PubMed

    Knowlden, Zackery A G; Sant, Andrea J

    2016-06-22

    Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

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

    PubMed

    Liu, Huixin; Liu, Wei; Hou, Xuexia; Zhang, Lin; Hao, Qin; Wan, Kanglin

    2016-01-01

    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.

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

    PubMed Central

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

    2013-01-01

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

  4. Salmonella expressing a T-cell epitope from Sendai virus are able to induce anti-infection immunity.

    PubMed

    Huang, He; Wang, Ye-Jun; White, Aaron P; Meng, Jia-Zi; Liu, Gui-Rong; Liu, Shu-Lin; Wang, Yue-Dan

    2009-09-01

    Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica subsp. enterica serovar Typhimurium LT2. DNA encoding an epitope from Sendai virus, SV9 (Sendai virus nucleoprotein peptide 324-332, FAPGNYPAL), which is known to induce cytotoxic T lymphocytes, was incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length DNA segment. To improve cytotoxic T lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) or arginine (RR) residues. Western blotting and immunofluorescence microscopy using AgfA-specific antiserum verified the expression of chimeric AgfA; expression was also proved by a Congo red binding assay. Oral immunizations of C57BL/6 mice with the four strains induced an epitope-specific T-cell response (detected by enzyme-linked immunosorbent spot assay). When the mice were challenged with the Sendai virus, the magnitude of the infection was significantly reduced in the immunized groups compared with the controls. The Salmonella fimbrial display system efficiently induces a cellular immune response and anti-infection immunity in vivo, providing a new strategy for the development of efficient peptide vaccination.

  5. The Challenges and Opportunities for Development of a T-Cell Epitope-Based Herpes Simplex Vaccine

    PubMed Central

    Kuo, Tiffany; Wang, Christine; Badakhshan, Tina; Chilukuri, Sravya; BenMohamed, Lbachir

    2014-01-01

    The infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) have been prevalent since the ancient Greek times. To this day, they still affect a staggering number of over a half billion individuals worldwide. HSV-2 infections cause painful genital herpes, encephalitis, and death in newborns. HSV-1 infections are more prevalent than HSV-2 infections and cause potentially blinding ocular herpes, oro-facial herpes and encephalitis. While genital herpes in mainly caused by HSV-2 infections, in recent years, there is an increase in the proportion of genital herpes caused by HSV-1 infections in young adults, which reach 50% in some western societies. While prophylactic and therapeutic HSV vaccines remain urgently needed for centuries their development has been notoriously difficult. During the most recent National Institute of Health (NIH) workshop titled "Next Generation Herpes Simplex Virus Vaccines: The Challenges and Opportunities", basic researchers, funding agencies, and pharmaceutical representatives gathered: (i) to assess the status of herpes vaccine research; and (ii) to identify the gaps and propose alternative approaches in developing a safe and efficient herpes vaccine. One “common denominator” among previously failed clinical herpes vaccine trials is that they either used a whole virus or whole viral proteins, which contain both pathogenic “symptomatic” and protective “asymptomatic” antigens/epitopes. In this report, we continue to advocate that using an “asymptomatic” epitope-based vaccine strategy that selectively incorporates protective epitopes which: (i) are exclusively recognized, in vitro, by effector memory CD4+ and CD8+ TEM cells from “naturally” protected seropositive asymptomatic individuals; and (ii) protect, in vivo, human leukocyte antigen (HLA) transgenic animal models from ocular and genital herpes infections and diseases, could be the answer to many of the scientific challenges facing HSV vaccine

  6. Pleiotropic effects of post-translational modifications on the fate of viral glycopeptides as cytotoxic T cell epitopes.

    PubMed

    Hudrisier, D; Riond, J; Mazarguil, H; Gairin, J E

    2001-10-12

    The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392-400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92-101 antigen (CSANNSHHYI). Both GP392-400 and GP92-101 bear a glycosylation motif, are naturally N-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2D(b) molecules, and are immunogenic. However, post-translational modifications differentially affected GP92-101 and GP392-400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392-400 but not for GP92-101. Further, under its N-glycosylated or de-N-glycosylated form, GP392-400 then lost its initial ability to generate a CTL response in mice, whereas GP92-101 was still immunogenic under the same conditions. The genetically encoded form of GP392-400, which on the basis of its immunogenicity could still be presented with H-2D(b) during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes.

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

    PubMed Central

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

    2007-01-01

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

  8. Identification of B-Cell Epitope of Dengue Virus Type 1 and Its Application in Diagnosis of Patients

    PubMed Central

    Wu, Han-Chung; Huang, Yue-Ling; Chao, Ting-Ting; Jan, Jia-Tsrong; Huang, Jau-Ling; Chiang, Hsien-Yuan; King, Chwan-Chuen; Shaio, Men-Fang

    2001-01-01

    Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine111 (His111) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine111 (Leu111). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1. PMID:11230414

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed Central

    2011-01-01

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

  11. Therapeutic Vaccination against the Rhesus Lymphocryptovirus EBNA-1 Homologue, rhEBNA-1, Elicits T Cell Responses to Novel Epitopes in Rhesus Macaques

    PubMed Central

    Silveira, Eduardo L. V.; Fogg, Mark H.; Leskowitz, Rachel M.; Ertl, Hildegund C.; Wiseman, Roger W.; O'Connor, David H.; Lieberman, Paul; Wang, Fred

    2013-01-01

    Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC–rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination. PMID:24089556

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

    PubMed Central

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

    2015-01-01

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

  13. Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

    PubMed Central

    Holtappels, Rafaela; Lemmermann, Niels A. W.; Podlech, Jürgen; Ebert, Stefan; Reddehase, Matthias J.

    2016-01-01

    Successful reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8+ T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8+ T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8+ T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as “immunodominant” epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a “personalized immunotherapy.” It is, therefore, an important question if IDE-specific CD8+ T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8+ T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8+ T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for human

  14. Immunodominant T-Cell Epitopes in the VP1 Capsid Protein of Rhinovirus Species A and C

    PubMed Central

    Gaido, Cibele M.; Stone, Shane; Chopra, Abha; Thomas, Wayne R.; Le Souëf, Peter N.

    2016-01-01

    ABSTRACT Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide, and RV-C has been linked to more severe exacerbations of asthma in young children. Little is known about the immune responses to the different RV species, although studies comparing IgG1 antibody titers found impaired antibody responses to RV-C. Therefore, the aim of this study was to assess whether T-cell immunity to RV-C is similarly impaired. We measured T-cell proliferation to overlapping synthetic peptides covering the entire VP1 capsid protein of an RV-A and RV-C genotype for 20 healthy adult donors. Human leukocyte antigen (HLA) was typed in all the donors in order to investigate possible associations between the HLA type and RV peptide recognition. Total and specific IgG1 antibody titers to the VP1 proteins of both RV-A and RV-C were also measured to examine associations between the antibody and T-cell responses. We identified T-cell epitopes that are specific to and representative of each RV-A and RV-C species. These epitopes stimulated CD4+-specific T-cell proliferation, with similar magnitudes of response for both RV species. All the donors, independent of their HLA-DR or -DQ type, were able to recognize the immunodominant RV-A and -C regions of VP1. Furthermore, the presence or absence of specific antibody titers was not related to changes in T-cell recognition. Our results indicate a dissociation between the antibody and T-cell responses to rhinoviruses. The species-representative T-cell epitopes identified in this study are valuable tools for future studies investigating T-cell responses to the different RV species. IMPORTANCE Rhinoviruses (RVs) are mostly associated with the common cold and asthma exacerbations, although their contributions to most upper and lower respiratory tract diseases have increasingly been reported. Species C (RV-C) has been associated with more frequent and severe asthma exacerbations in young children and, along with

  15. Stem cell-mediated gene delivering for the treatment of cerebral ischemia: progress and prospectives.

    PubMed

    Chen, Chao; Wang, Yongting; Yang, Guo-Yuan

    2013-01-01

    Ischemic stroke is one of the leading causes of death and disability worldwide. There is no effective treatment for ischemic stroke apart from thrombolytic therapy, which has a narrow therapeutic time window. Gene therapy has proven to be effective in experimental stroke, but it suffers from disadvantages that limit its clinical application, such as difficulty in intracranial delivering of therapeutic genes, low efficacy in transfecting host cells and long-term expression of exogenous genes. Delivering therapeutic genes to the ischemic brain via stem cells is an alternative strategy of combined gene and stem cell therapy. There are advantages for stem cell-mediated gene delivery as opposed to direct gene transfer. In recent years, studies used stem cells that over-express different neurotrophic factors, such as BDNF, GDNT, or NT3, and found that the delivery of these genetically-modified stem cells to animal models of ischemic stroke is safe and effective, thus suggesting that stem cell-based gene therapy may be a promising treatment for stroke. This review summarizes the advantages and recent progress of stem cell-based gene therapy for ischemic stroke. We also discuss the relevant strategy for optimizing stem cell-based gene therapy and discuss the potential strategies for its future application.

  16. Automatic Generation of Validated Specific Epitope Sets.

    PubMed

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

    2015-01-01

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

  17. Phase I active immunotherapy with combination of two chimeric, human epidermal growth factor receptor 2, B-cell epitopes fused to a promiscuous T-cell epitope in patients with metastatic and/or recurrent solid tumors.

    PubMed

    Kaumaya, Pravin T P; Foy, Kevin Chu; Garrett, Joan; Rawale, Sharad V; Vicari, Daniele; Thurmond, Jennifer M; Lamb, Tammy; Mani, Aruna; Kane, Yahaira; Balint, Catherine R; Chalupa, Donald; Otterson, Gregory A; Shapiro, Charles L; Fowler, Jeffrey M; Grever, Michael R; Bekaii-Saab, Tanios S; Carson, William E

    2009-11-01

    PURPOSE To evaluate the maximum-tolerated dose (MTD), safety profile, and immunogenicity of two chimeric, B-cell epitopes derived from the human epidermal growth factor receptor (HER2) extracellular domain in a combination vaccine with a promiscuous T-cell epitope (ie, MVF) and nor-muramyl-dipeptide as adjuvant emulsified in SEPPIC ISA 720. PATIENTS AND METHODS Eligible patients with metastatic and/or recurrent solid tumors received three inoculations on days 1, 22, and 43 at doses of total peptide that ranged from 0.5 to 3.0 mg. Immunogenicity was evaluated by enzyme-linked immunosorbent assay, flow cytometry, and HER2 signaling assays. Results Twenty-four patients received three inoculations at the intended dose levels, which elicited antibodies able to recognize native HER2 receptor and inhibited both the proliferation of HER2-expressing cell lines and phosphorylation of the HER2 protein. The MTD was determined to be the highest dose level of 3.0 mg of the combination vaccine. There was a significant increase from dose level 1 (0.5 mg) to dose level 4 (3.0 mg) in HER2-specific antibodies. Four patients (one each with adrenal, colon, ovarian, and squamous cell carcinoma of unknown primary) were judged to have stable disease; two patients (one each with endometrial and ovarian cancer) had partial responses; and 11 patients had progressive disease. Patients with stable disease received 6-month boosts, and one patient received a 20-month boost. CONCLUSION The combination vaccines were safe and effective in eliciting antibody responses in a subset of patients (62.5%) and were associated with no serious adverse events, autoimmune disease, or cardiotoxicity. There was preliminary evidence of clinical activity in several patients.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2014-01-01

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

  20. Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden.

    PubMed

    Sanecka, Anna; Yoshida, Nagisa; Dougan, Stephanie K; Jackson, John; Shastri, Nilabh; Ploegh, Hidde; Blanchard, Nicolas; Frickel, Eva-Maria

    2016-11-01

    We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the L(d) -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound L(d) -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

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

    PubMed Central

    2012-01-01

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

  2. A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses.

    PubMed

    Takagi, Hidenori; Hiroi, Takachika; Yang, Lijun; Tada, Yoshifumi; Yuki, Yoshikazu; Takamura, Kaoru; Ishimitsu, Ryotaro; Kawauchi, Hideyuki; Kiyono, Hiroshi; Takaiwa, Fumio

    2005-11-29

    Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. Under the control of the rice seed storage protein glutelin GluB-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein. Oral feeding to mice of transgenic rice seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4(+) T cell proliferative responses. The levels of allergen-specific CD4(+) T cell-derived allergy-associated T helper 2 cytokine production of IL-4, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens.

  3. H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance

    PubMed Central

    Liu, Rui; Moise, Leonard; Tassone, Ryan; Gutierrez, Andres H; Terry, Frances E; Sangare, Kotou; Ardito, Matthew T; Martin, William D; De Groot, Anne S

    2015-01-01

    Avian-origin H7N9 influenza is a novel influenza A virus (IAV) that emerged in humans in China in 2013. Using immunoinformatics tools, we identified several H7N9 T cell epitopes with T cell receptor (TCR)-facing residues identical to those of multiple epitopes from human proteins. We hypothesized that host tolerance to these peptides may impair T helper response and contribute to the low titer, weak hemagglutination inhibiting (HI) antibody responses and diminished seroconversion rates that have been observed in human H7N9 infections and vaccine trials. We found that the magnitude of human T effector responses to individual H7N9 peptides was inversely correlated with the peptide's resemblance to self. Furthermore, a promiscuous T cell epitope from the hemagglutinin (HA) protein suppressed responses to other H7N9 peptides when co-administered in vitro. Along with other highly ‘human-like’ peptides from H7N9, this peptide was also shown to expand FoxP3+ regulatory T cells (Tregs). Thus, H7N9 may be camouflaged from effective human immune response by T cell epitope sequences that avert or regulate effector T cell responses through host tolerance. PMID:26090577

  4. Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells.

    PubMed

    Ríos-Huerta, Regina; Monreal-Escalante, Elizabeth; Govea-Alonso, Dania O; Angulo, Carlos; Rosales-Mendoza, Sergio

    2017-02-01

    An antigenic protein targeting two epitopes from the Zaire ebolavirus GP1 protein was expressed in plant cells rendering an antigen capable of inducing humoral responses in mouse when administered subcutaneously or orally. The 2014 Ebola outbreak made clear that new treatments and prophylactic strategies to fight this disease are needed. Since vaccination is an intervention that could achieve the control of this epidemic disease, exploring the production of new low-cost vaccines is a key path to consider; especially in developing countries. In this context, plants are attractive organisms for the synthesis and delivery of subunit vaccines. This study aimed at producing a chimeric protein named LTB-EBOV, based on the B subunit of the Escherichia coli heat-labile enterotoxin as an immunogenic carrier and two epitopes from the Zaire ebolavirus GP1 protein recognized by neutralizing antibodies. The LTB-EBOV protein was expressed in plant tissues at levels up to 14.7 µg/g fresh leaf tissue and proven to be immunogenic in BALB/c mice when administered by either subcutaneous or oral routes. Importantly, IgA and IgG responses were induced following the oral immunization. The potential use of the plant-made LTB-EBOV protein against EBOV is discussed.

  5. A novel B cell epitope in cold-shock DEAD-box protein A from Mycobacterium tuberculosis.

    PubMed

    Wang, Huanan; Zhu, Ting; Yu, Shenye; Liu, Huifang; Wang, Xiumei; Chen, Liping; Si, Wei; Pang, Hai; Liu, Siguo

    2013-06-01

    In this study, a hybridoma-based technique and phage display technology were used to obtain mouse monoclonal antibodies (mAb) against cold-shock DEAD-box protein A (CsdA) from Mycobacterium tuberculosis and to determine the location of the relevant epitope. One highly specific mAb, named A3G5, was developed against the recombinant CsdA protein (rCsdA) and could detect rCsdA protein in enzyme-linked immunosorbent assays (ELISA) and Western blot assays. By screening a phage displayed library of random 12-mers (Ph.D.-12), 10 positive phage clones were randomly selected after three rounds of bio-panning and identified by ELISA. Eight of these clones were sequenced, and their amino acid sequences were deduced. One B-cell epitope (-APDPPLSRR-) in the rCsdA protein was identified with mAb A3G5. A synthetic peptide (-MAPDPPLSRR-) (Cpep) matched well with the CsdA sequence at 443-451 aa and was confirmed by affinity ELISA, competitive inhibition assays and the development of an immune response in mice. These results may be of great potential value in the further analysis of the function and structure of the CsdA protein from M. tuberculosis.

  6. A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

    PubMed Central

    Chan, Kok-Fei; Oveissi, Sara; Jackson, Heather M.; Dimopoulos, Nektaria; Guillaume, Philippe; Knights, Ashley J.; Lowen, Tamara; Robson, Neil C.; Russell, Sarah E.; Scotet, Emmanuel; Davis, Ian D.; Maraskovsky, Eugene; Cebon, Jonathan; Luescher, Immanuel F.; Chen, Weisan

    2012-01-01

    NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. PMID:22970293

  7. A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

    PubMed Central

    Wang, Si; Placzek, William J.; Stebbins, John L.; Mitra, Sayantan; Noberini, Roberta; Koolpe, Mitchell; Zhang, Ziming; Dahl, Russell; Pasquale, Elena B.; Pellecchia, Maurizio

    2012-01-01

    The efficacy of anti-cancer drugs is often limited by their systemic toxicities and adverse side effects. We report that the EphA2 receptor is over-expressed preferentially in several human cancer cell lines compared to normal tissues and that an EphA2 targeting peptide (YSAYPDSVPMMS) can be effective in delivering anti-cancer agents to such tumors. Hence, we report on the synthesis and characterizations of a novel EphA2-targeting agent conjugated with the chemotherapeutic drug paclitaxel. We found that the peptide-drug conjugate is dramatically more effective than paclitaxel alone at inhibiting tumor growth in a prostate cancer xenograft model, delivering significantly higher levels of drug to the tumor site. We believe these studies open the way to the development of a new class of therapeutic compounds that exploit the EphA2 receptor for drug delivery to cancer cells. PMID:22329578

  8. Novel epitope evoking CD138 antigen-specific cytotoxic T lymphocytes targeting multiple myeloma and other plasma cell disorders

    PubMed Central

    Bae, Jooeun; Tai, Yu-Tzu; Anderson, Kenneth C.; Munshi, Nikhil C.

    2012-01-01

    The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138260-268 (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138+ MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8+ activated/memory T cells with a low percentage of CD4+ T cell and naive CD8+ T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ–interferon secretion to HLA-A2+/CD138+ myeloma cells, but not HLA-A2−/CD138+ or HLA-A2+/CD138− cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2+/CD138+ cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138260-268 (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies. PMID:21902685

  9. Enhanced potency of cell-based therapy for ischemic tissue repair using an injectable bioactive epitope presenting nanofiber support matrix.

    PubMed

    Tongers, Jörn; Webber, Matthew J; Vaughan, Erin E; Sleep, Eduard; Renault, Marie-Ange; Roncalli, Jerome G; Klyachko, Ekaterina; Thorne, Tina; Yu, Yang; Marquardt, Katja-Theres; Kamide, Christine E; Ito, Aiko; Misener, Sol; Millay, Meredith; Liu, Ting; Jujo, Kentaro; Qin, Gangjian; Losordo, Douglas W; Stupp, Samuel I; Kishore, Raj

    2014-09-01

    The translation of cell-based therapies for ischemic tissue repair remains limited by several factors, including poor cell survival and limited target site retention. Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cell-based therapies. Given the relevance of integrin signaling for cellular homeostasis, we developed an injectable, bioactive peptide-based nanofiber matrix that presents an integrin-binding epitope derived from fibronectin, and evaluated its feasibility as a supportive artificial matrix for bone marrow-derived pro-angiogenic cells (BMPACs) used as a therapy in ischemic tissue repair. Incubation of BMPACs with these peptide nanofibers in vitro significantly attenuated apoptosis while enhancing proliferation and adhesion. Pro-angiogenic function was enhanced, as cells readily formed tubes. These effects were, in part, mediated via p38, and p44/p42 MAP kinases, which are downstream pathways of focal adhesion kinase. In a murine model of hind limb ischemia, an intramuscular injection of BMPACs within this bioactive peptide nanofiber matrix resulted in greater retention of cells, enhanced capillary density, increased limb perfusion, reduced necrosis/amputation, and preserved function of the ischemic limb compared to treatment with cells alone. This self-assembling, bioactive peptide nanofiber matrix presenting an integrin-binding domain of fibronectin improves regenerative efficacy of cell-based strategies in ischemic tissue by enhancing cell survival, retention, and reparative functions.

  10. In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

    PubMed Central

    Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Singh, Balwan; Jiang, Jianlin; Meyer, Esmeralda V. S.; Santos, Fátima; Banic, Dalma Maria; Moreno, Alberto; Galinski, Mary R.; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa

    2016-01-01

    Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and

  11. Soft electroporation for delivering molecules into tightly adherent mammalian cells through 3D hollow nanoelectrodes.

    PubMed

    Caprettini, Valeria; Cerea, Andrea; Melle, Giovanni; Lovato, Laura; Capozza, Rosario; Huang, Jian-An; Tantussi, Francesco; Dipalo, Michele; De Angelis, Francesco

    2017-08-17

    Electroporation of in-vitro cultured cells is widely used in biological and medical areas to deliver molecules of interest inside cells. Since very high electric fields are required to electroporate the plasma membrane, depending on the geometry of the electrodes the required voltages can be very high and often critical to cell viability. Furthermore, in traditional electroporation configuration based on planar electrodes there is no a priori certain feedback about which cell has been targeted and delivered and the addition of fluorophores may be needed to gain this information. In this study we present a nanofabricated platform able to perform intracellular delivery of membrane-impermeable molecules by opening transient nanopores into the lipid membrane of adherent cells with high spatial precision and with the application of low voltages (1.5-2 V). This result is obtained by exploiting the tight seal that the cells present with 3D fluidic hollow gold-coated nanostructures that act as nanochannels and nanoelectrodes at the same time. The final soft-electroporation platform provides an accessible approach for controlled and selective drug delivery on ordered arrangements of cells.

  12. Immune response characterization and vaccine potential of a recombinant chimera comprising B-cell epitope of Aeromonas hydrophila outer membrane protein C and LTB.

    PubMed

    Sharma, Mahima; Dixit, Aparna

    2016-12-07

    Aeromonas hydrophila is one of the most virulent fish pathogens, causing colossal economic losses to the aquaculture industry annually. The absence of a safe and effective vaccine makes it very difficult to control this infection. Outer membrane proteins have been widely illustrated to confer protective immunity against a broad spectrum of gram negative bacteria. In the current study, we have analyzed the ability of B-cell epitopes of A. hydrophila's outer membrane protein C (OmpC) to confer protection against bacterial virulence. Bioinformatic algorithms were used to predict linear B-cell epitopes of OmpC and the corresponding nucleotide sequences were cloned in translational fusion with heat labile enterotoxin B subunit (LTB) of E. coli. Of the three recombinant LTB.epitope fusion proteins evaluated, antisera against the fusion protein comprising the epitope stretch of 143-175 amino acids gave maximum cross reactivity with the parent protein OmpC. The anti-fusion protein antisera contained both OmpC- and LTB-specific antibodies. The fusion proteins' LTB moiety retained its ability to bind to the GM1 ganglioside receptor, an essential requirement for its adjuvanicity. Antibody isotyping, cytokine ELISA, and cytokine array analysis revealed a Th2 skewed type immune response along with the presence of some relevant Th17 and Th1 cytokines involved in conferring protective immunity. Surface exposure of the epitope143-175 on live A. hydrophila membrane was investigated and validated using bacterial agglutination and flow cytometry analysis using anti-fusion protein antisera. Our results strongly support the potential of B-cell epitope143-175 of OmpC of A. hydrophila, in fusion with the LTB, as an effective and promising vaccine candidate against this bacterium. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. T-cell epitope strength in WAP-T mouse mammary carcinomas is an important determinant in PD1/PD-L1 immune checkpoint blockade therapy

    PubMed Central

    Bruns, Michael; Wanger, Jara; Schumacher, Udo; Deppert, Wolfgang

    2016-01-01

    Using the SV40 transgenic WAP-T/WAP-TNP mouse models for mammary carcinomas, we compared the response to immune checkpoint blockade therapy in tumor mice expressing either SV40 T-antigen containing the LCMV NP-epitope (T-AgNP in WAP-TNP mice), or the unmodified T-antigen (T-Ag in WAP-T mice). Specifically, we asked, whether the presence of the highly immunogenic NP-epitope in T-AgNP influences this response in comparison to the weakly immunogenic T-cell epitopes of T-Ag in WAP-T tumor mice. Treatment of WAP-TNP tumor mice with either anti-PD1 or anti-PD-L1 antibodies led to tumor regression, with anti-PD-L1 treatment being more effective. However, tumors had fully re-appeared after 21 days, indicating that CTL exhaustion had been rapidly re-established. Surprisingly, the same treatment applied to WAP-T tumor mice resulted in a significantly prolonged period of tumor regression. We provide evidence that in contrast to the weak antigenic stimuli exerted by T-cell epitopes of T-Ag, the strong antigenic stimulus of the NP-epitope in T-AgNP has a dual effect: (i) a rapid generation of active NP-specific CTLs, accompanied (ii) by accelerated CTL exhaustion. Our data support the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy. PMID:27579535

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

    PubMed

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

    2012-11-01

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

  15. Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal.

    PubMed

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Bucris, Efrat; Ziv-Av, Amotz; Xiang, Cunli; Bobbitt, Kevin; Rempel, Sandra A; Hasselbach, Laura; Mikkelsen, Tom; Slavin, Shimon; Brodie, Chaya

    2013-02-01

    MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.

  16. Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal

    PubMed Central

    Lee, Hae Kyung; Finniss, Susan; Cazacu, Simona; Bucris, Efrat; Ziv-Av, Amotz; Xiang, Cunli; Bobbitt, Kevin; Rempel, Sandra A.; Hasselbach, Laura; Mikkelsen, Tom; Slavin, Shimon; Brodie, Chaya

    2013-01-01

    MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction–dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo. PMID:23548312

  17. Mapping IgE-binding epitopes of Ric c 1 and Ric c 3, allergens from Ricinus communis, by mast cell degranulation assay.

    PubMed

    Felix, S P; Mayerhoffer, R O; Damatta, R A; Verícimo, M A; Nascimento, V V; Machado, O L T

    2008-04-01

    Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.

  18. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice

    PubMed Central

    Pardy, Ryan D.; Rajah, Maaran M.; Taylor, Nathan G.

    2017-01-01

    Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies. PMID:28231312

  19. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

    PubMed

    Pardy, Ryan D; Rajah, Maaran M; Condotta, Stephanie A; Taylor, Nathan G; Sagan, Selena M; Richer, Martin J

    2017-02-01

    Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.

  20. Producer T cells: Using genetically engineered T cells as vehicles to generate and deliver therapeutics to tumors

    PubMed Central

    Tsai, Alexander K.; Davila, Eduardo

    2016-01-01

    ABSTRACT Adoptive cell transfer (ACT) is an emerging anticancer therapy that has shown promise in various malignancies. Redirecting antigen specificity by genetically engineering T cells to stably express receptors has become an effective variant of ACT. A novel extension of this approach is to utilize engineered T cells to produce and deliver anticancer therapeutics that enhance cytotoxic T cell function and simultaneously inhibit immunosuppressive processes. Here, we review the potential of using T cells as therapeutic-secreting vehicles for immunotherapies and present theoretical and established arguments in support of further development of this unique cell-based immunotherapy. PMID:27467930

  1. Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35.

    PubMed

    Jung, Mi-Jeong; Moon, Young-Chan; Cho, Ik-Hyun; Yeh, Jung-Yong; Kim, Sun-Eui; Chang, Wha-Seok; Park, Seung-Young; Song, Chang-Seon; Kim, Hwi-Yool; Park, Keun-Kyu; McOrist, Steven; Choi, In-Soo; Lee, Joong-Bok

    2005-03-01

    Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.

  2. Genetic linkage of autologous T cell epitopes in a chimeric recombinant construct improves anti-parasite and anti-disease protective effect of a malaria vaccine candidate.

    PubMed

    Singh, Balwan; Cabrera-Mora, Monica; Jiang, Jianlin; Galinski, Mary; Moreno, Alberto

    2010-03-19

    We have reported the design of polyvalent synthetic and recombinant chimeras that include promiscuous T cell epitopes as a viable delivery system for pre-erythrocytic subunit malaria vaccines. To further assess the ability of several Plasmodium T cell epitopes to enhance vaccine potency, we designed a synthetic gene encoding four Pla