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Sample records for cell epitope generated

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

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    Farzaneh Farzin

    2010-05-01

    Full Text Available Abstract 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

  2. The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy

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    Kessler, Jan

    2009-01-01

    Cytotoxic T cell epitopes are the targets for a T cell mediated immunotherapy of cancer. The thesis reports on their identification in the tumor associated proteins BCR-ABL and PRAME by the reverse immunology (prediction) strategy. An extended strategy is used, including the analysis of the

  3. Automatic Generation of Validated Specific Epitope Sets

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    Sebastian Carrasco Pro

    2015-01-01

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

  4. Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC.

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    Holen, E; Elsayed, S

    1996-09-01

    Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.

  5. Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

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    Sahoo, Malaya K.; Tan, Susanna K.; Chen, Sharon F.; Kapusinszky, Beatrix; Concepcion, Katherine R.; Kjelson, Lynn; Mallempati, Kalyan; Farina, Heidi M.; Fernández-Viña, Marcelo; Tyan, Dolly; Grimm, Paul C.; Anderson, Matthew W.; Concepcion, Waldo

    2015-01-01

    BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies. PMID:26202116

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

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    Sabrina de Almeida Lima

    2018-04-01

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

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitop...... that is specifically associated with a plant cell separation process that results in complete cell detachment....... is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically...... associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified...

  8. A broadly applicable approach to T cell epitope identification: application to improving tumor associated epitopes and identifying epitopes in complex pathogens.

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    Valentino, Michael D; Abdul-Alim, C Siddiq; Maben, Zachary J; Skrombolas, Denise; Hensley, Lucinda L; Kawula, Thomas H; Dziejman, Michelle; Lord, Edith M; Frelinger, Jeffrey A; Frelinger, John G

    2011-10-28

    Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. High frequency of T cells specific for cryptic epitopes in melanoma patients

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    Andersen, Rikke Sick; Andersen, Sofie Ramskov; Hjortsø, Mads Duus

    2013-01-01

    A number of cytotoxic T-cell epitopes are cryptic epitopes generated from non-conventional sources. These include epitopes that are encoded by alternative open reading frames or in generally non-coding genomic regions, such as introns. We have previously observed a frequent recognition of cryptic...... epitopes by tumor infiltrating lymphocytes isolated from melanoma patients. Here, we show that such cryptic epitopes are more frequently recognized than antigens of the same class encoded by canonical reading frames. Furthermore, we report the presence of T cells specific for three cryptic epitopes encoded...

  10. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

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    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  11. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

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    Flego, Michela; Ascione, Alessandro; Zamboni, Silvia; Dupuis, Maria L; Imperiale, Valentina; Cianfriglia, Maurizio

    2007-01-01

    Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP). Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease. PMID:17605808

  12. Improved method for predicting linear B-cell epitopes

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    Larsen, Jens Erik Pontoppidan; Lund, Ole; Nielsen, Morten

    2006-01-01

    Background B-cell epitopes are the sites of molecules that are recognized by antibodies of the immune system. Knowledge of B-cell epitopes may be used in the design of vaccines and diagnostics tests. It is therefore of interest to develop improved methods for predicting B-cell epitopes. In this paper, we describe an improved method for predicting linear B-cell epitopes. Results In order to do this, three data sets of linear B-cell epitope annotated proteins were constructed. A data set was co...

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

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    Kirsten E Wiens

    Full Text Available 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.

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

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    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.

  15. Artificial intelligence methods for predicting T-cell epitopes.

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    Zhao, Yingdong; Sung, Myong-Hee; Simon, Richard

    2007-01-01

    Identifying epitopes that elicit a major histocompatibility complex (MHC)-restricted T-cell response is critical for designing vaccines for infectious diseases and cancers. We have applied two artificial intelligence approaches to build models for predicting T-cell epitopes. We developed a support vector machine to predict T-cell epitopes for an MHC class I-restricted T-cell clone (TCC) using synthesized peptide data. For predicting T-cell epitopes for an MHC class II-restricted TCC, we built a shift model that integrated MHC-binding data and data from T-cell proliferation assay against a combinatorial library of peptide mixtures.

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

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    Guan Pingping

    2006-03-01

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

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

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    Almut Scherer

    2006-08-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    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......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...... and immunology community....

  19. Recent advances in B-cell epitope prediction methods

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    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

  20. Predicting linear B-cell epitopes using string kernels

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    EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

    2008-01-01

    The identification and characterization of B-cell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting linear B-cell epitopes are highly desirable. We evaluated Support Vector Machine (SVM) classifiers trained utilizing five different kernel methods using fivefold cross-validation on a homology-reduced data set of 701 linear B-cell epitopes, extracted from Bcipep database, and 701 non-epitopes, randomly extracted from SwissProt sequences. Based on the results of our computational experiments, we propose BCPred, a novel method for predicting linear B-cell epitopes using the subsequence kernel. We show that the predictive performance of BCPred (AUC = 0.758) outperforms 11 SVM-based classifiers developed and evaluated in our experiments as well as our implementation of AAP (AUC = 0.7), a recently proposed method for predicting linear B-cell epitopes using amino acid pair antigenicity. Furthermore, we compared BCPred with AAP and ABCPred, a method that uses recurrent neural networks, using two data sets of unique B-cell epitopes that had been previously used to evaluate ABCPred. Analysis of the data sets used and the results of this comparison show that conclusions about the relative performance of different B-cell epitope prediction methods drawn on the basis of experiments using data sets of unique B-cell epitopes are likely to yield overly optimistic estimates of performance of evaluated methods. This argues for the use of carefully homology-reduced data sets in comparing B-cell epitope prediction methods to avoid misleading conclusions about how different methods compare to each other. Our homology-reduced data set and implementations of BCPred as well as the APP method are publicly available through our web-based server, BCPREDS, at: http://ailab.cs.iastate.edu/bcpreds/. PMID:18496882

  1. Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1 Frame of U79260(FTO

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    Michael Linnebacher

    2010-01-01

    Full Text Available Microsatellite instability (MSI-H induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs. Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1 frame of U79260(FTO encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV. T cells specific for FSP11 efficiently recognized HLA-A0201(pos tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO in MSI-H colorectal carcinoma (81.8%, this recommends that FSP11 be a component of future vaccines.

  2. The polyclonal CD8 T cell response to influenza M158-66 generates a fully connected network of cross-reactive clonotypes to structurally related peptides: a paradigm for memory repertoire coverage of novel epitopes or escape mutants.

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    Petrova, Galina V; Naumova, Elena N; Gorski, Jack

    2011-06-01

    Cross-reactivity of T cells is defined as recognition of two or more peptide-MHC complexes by the same T cell. Although examples of cross-reactivity have been reported, a detailed examination of cross-reactivity has not been performed. In this study, we took advantage of the high degree of polyclonality in the BV19 T cell repertoire responding to influenza M1(58-66) in HLA-A2 individuals to obtain a measure of simple cross-reactivity. We used substitutions that incrementally change the structure of the M1(58-66) peptide to measure how the HLA-A2-restricted response adapts to these changes. In three HLA-A2 adult subjects, we identified the BV19 clonotypes in the recall response to the influenza epitope M1(58-66) and 12 M1 peptides substituted at TCR contact position 63 or 65. The fraction of cross-reactive clonotypes in the M1(58-66) repertoire varied from 45-58% in the three donors. The extent of cross-reactivity, which is the additional number of peptides recognized by a single clonotype, is as high as six. We summarized the data using graph theory, with the cross-reactive clonotypes connecting the different HLA-A2 peptides recognized. The cross-reactive clonotypes form a well-connected network that could provide protection from virus-escape variants. We predict that any new pathogen with an epitope whose shape corresponds to that of the peptides that we studied would find a pre-existing repertoire ready to respond to it. We propose that in adult memory repertoires, previously encountered epitopes may have generated similar cross-reactive repertoires.

  3. Analysis of cytotoxic T cell epitopes in relation to cancer

    DEFF Research Database (Denmark)

    Stranzl, Thomas

    kill the infected cells. The focus of my PhD project has been on improving a method for CTL epitope pathway prediction, on analyzing the epitope density in the alternative cancer exome, and on a study investigating minor histocompatibility antigens (mHags) associated with leukemia. Part I......The human immune system is a highly adaptable system, defending our bodies against pathogens and tumor cells. Cytotoxic T cells (CTL) are cells of the adaptive immune system, capable of inducing a programmed cell death and thus able to eliminate infected or tumor cells. CTLs discriminate between...... healthy and infected cells based on peptide fragments presented on the cells surface. All nucleated cells present these peptide fragments in complex with Major Histocompatibility Complex (MHC) class I molecules. Peptides that are recognized by CTLs are called epitopes and induce the CTLs to subsequently...

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

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

    1999-08-15

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

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

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    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinfor......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...... 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....

  7. Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate.

    Science.gov (United States)

    Salvat, Regina S; Parker, Andrew S; Guilliams, Andrew; Choi, Yoonjoo; Bailey-Kellogg, Chris; Griswold, Karl E

    2014-12-01

    Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed "T cell epitopes." Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure-function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4-5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.

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

    Directory of Open Access Journals (Sweden)

    Dimitrios eTsiantoulas

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Veronika I Zarnitsyna

    2016-06-01

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

  10. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

    Directory of Open Access Journals (Sweden)

    Pedersen Henriette L

    2008-05-01

    Full Text Available Abstract Background Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Results Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15 to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. Conclusion These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell

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

    Directory of Open Access Journals (Sweden)

    Christopher Chiu

    2014-03-01

    Full Text Available Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV, we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1 along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

    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.

  14. Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Jinfeng Ti

    Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

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

    Science.gov (United States)

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

    2015-01-01

    Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Accurate prediction of immunogenic T-cell epitopes from epitope sequences using the genetic algorithm-based ensemble learning.

    Science.gov (United States)

    Zhang, Wen; Niu, Yanqing; Zou, Hua; Luo, Longqiang; Liu, Qianchao; Wu, Weijian

    2015-01-01

    T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system. In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set. Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant. The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.

  17. Accurate prediction of immunogenic T-cell epitopes from epitope sequences using the genetic algorithm-based ensemble learning.

    Directory of Open Access Journals (Sweden)

    Wen Zhang

    Full Text Available T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system.In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve on the training set.Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant.The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

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

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

    Science.gov (United States)

    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.

  1. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3).

    Science.gov (United States)

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

    2009-10-13

    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. 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-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. 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.

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

    Science.gov (United States)

    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.

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

    African Journals Online (AJOL)

    AJL

    2012-05-01

    May 1, 2012 ... Outer membrane protein U (OmpU), an adhesion protein of Vibrio mimicus, is a good antigen, but its epitopes are still unclear. In order to locate the epitopes of OmpU protein, epitope prediction was performed using the amino acid sequence of OmpU protein of V. mimicus HX4 strain that was isolated.

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

    African Journals Online (AJOL)

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

  5. Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope

    International Nuclear Information System (INIS)

    Ciernik, Ilja F.; Romero, Pedro; Berzofsky, Jay A.; Carbone, David P.

    1999-01-01

    Background: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. Methods: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. Results: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. Conclusions: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines

  6. Immune epitope database analysis resource

    DEFF Research Database (Denmark)

    Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang

    2012-01-01

    The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide...

  7. Identification of a conserved linear B-cell epitope in the M protein of porcine epidemic diarrhea virus

    Directory of Open Access Journals (Sweden)

    Zhang Zhibang

    2012-10-01

    Full Text Available Abstract Background The major structural protein of coronaviruses, the membrane (M protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV. Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein. Results One hybridoma cell line secreting anti-M protein monoclonal antibody (McAb was generated and designated 4D4. To map the epitopes on the PEDV M protein, a total of 17 partially overlapping fragments covering the C-terminus of M protein were expressed as fusion proteins with a 6×His tag or a GST tag. A linear motif, 193TGWAFYVR200, was identified by enzyme-linked immunosorbent assay (ELISA and western blot (WB analysis using McAb 4D4. The motif 195WAFYVR200 was the minimal requirement for reactivity, as demonstrated by removing amino acids individually from both ends of the motif 193TGWAFYVR200. The result of WB analysis showed that the 4D4-defined epitope could be recognized by PEDV-positive serum, but not transmissible gastroenteritis virus (TGEV-positive serum. Furthermore, this epitope was highly conserved among different PEDV strains, as shown by alignment and comparison of sequences. Conclusion A McAb, 4D4, directed against the M protein of PEDV, was obtained, and the 4D4-defined minimal epitope sequence was 195WAFYVR200. The McAb could serve as a candidate for development of a McAb-based antigen capture ELISA for detection of PEDV. The epitope identified provides a basis for the development of epitope-based differential diagnostic techniques and may be useful in the design of epitope-based vaccines.

  8. Conservation analysis of dengue virust-cell epitope-based vaccine candidates using peptide block entropy

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Zhang, Guang Lan; Keskin, Derin B.

    2011-01-01

    Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches are based on combinations of highly conserved T-cell epitopes. Peptide block entropy analysis is a novel approach for assembling sets of ...

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

    Indian Academy of Sciences (India)

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

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

    Indian Academy of Sciences (India)

    WINTEC

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

  11. Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

    Science.gov (United States)

    Sircar, Gaurab; Jana, Kuladip; Dasgupta, Angira; Saha, Sudipto; Gupta Bhattacharya, Swati

    2016-08-19

    Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing

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

    Science.gov (United States)

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

    2015-02-01

    Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. EPITOPE MAPPING OF SCLC-CLUSTER-2 MABS AND GENERATION OF ANTIBODIES DIRECTED AGAINST NEW EGP-2 EPITOPES

    NARCIS (Netherlands)

    HELFRICH, W; KONING, PW; THE, TH; DELEIJ, L

    1994-01-01

    Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it

  14. Superior control of HIV-1 replication by CD8+ T cells targeting conserved epitopes: implications for HIV vaccine design.

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    Pratima Kunwar

    Full Text Available A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i increasing the breadth of vaccine-induced responses or (ii increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8(+ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8(+ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS by three different methods (prevalence, entropy and conseq on clade-B and group-M sequence alignments. The majority of CD8(+ T cell responses were directed against variable epitopes (p<0.01. Interestingly, increasing breadth of CD8(+ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009. Moreover, subjects possessing CD8(+ T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021. The association between viral control and the breadth of conserved CD8(+ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215. The associations with viral control were independent of functional avidity of CD8(+ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus

  15. Conserved B-cell epitopes among human bocavirus species indicate potential diagnostic targets.

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    Zhuo Zhou

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

  16. Computer-aided design of T-cell epitope-based vaccines: addressing population coverage.

    Science.gov (United States)

    Oyarzun, P; Kobe, B

    2015-10-01

    Epitope-based vaccines (EVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant - immunogenic and conserved - antigen regions. Experimental screening of large sets of peptides is time-consuming and costly; therefore, in silico methods that facilitate T-cell epitope mapping of protein antigens are paramount for EV development. The prediction of T-cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T-cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T-cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state-of-the-art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens. © 2015 John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    1996-07-01

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

  18. The ADAMTS131239-1253peptide is a dominant HLA-DR1-restricted CD4+T-cell epitope.

    Science.gov (United States)

    Gilardin, Laurent; Delignat, Sandrine; Peyron, Ivan; Ing, Mathieu; Lone, Yu-Chun; Gangadharan, Bagirath; Michard, Baptiste; Kherabi, Yousra; Sharma, Meenu; Pashov, Anastas; Latouche, Jean-Baptiste; Hamieh, Mohamad; Toutirais, Olivier; Loiseau, Pascale; Galicier, Lionel; Veyradier, Agnès; Kaveri, Srini; Maillère, Bernard; Coppo, Paul; Lacroix-Desmazes, Sébastien

    2017-11-01

    Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13 th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4 + T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4 + T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4 + T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4 + T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS13 1239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4 + T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4 + T cells from Sure-L1 mice and was recognized by CD4 + T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS13 1239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4 + T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS13 1239-1253 -loaded HLA-DR tetramers. Copyright© Ferrata Storti Foundation.

  19. The ADAMTS131239–1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope

    Science.gov (United States)

    Gilardin, Laurent; Delignat, Sandrine; Peyron, Ivan; Ing, Mathieu; Lone, Yu-Chun; Gangadharan, Bagirath; Michard, Baptiste; Kherabi, Yousra; Sharma, Meenu; Pashov, Anastas; Latouche, Jean-Baptiste; Hamieh, Mohamad; Toutirais, Olivier; Loiseau, Pascale; Galicier, Lionel; Veyradier, Agnès; Kaveri, Srini; Maillère, Bernard; Coppo, Paul; Lacroix-Desmazes, Sébastien

    2017-01-01

    Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against “A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239–1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239–1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239–1253-loaded HLA-DR tetramers. PMID:28751567

  20. Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard

    2004-01-01

    a single dominant epitope may suppress the response to other viral epitopes, and this may lead to increased susceptibility to reinfection with escape variants circulating in the host population. To address these issues, we induced a memory response consisting solely of monospecific, CD8+ T cells by use...... of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral...... variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version...... 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...

  2. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

    DEFF Research Database (Denmark)

    Marcus, Susan E; Verhertbruggen, Yves; Hervé, Cécile

    2008-01-01

    BACKGROUND: Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally...... is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components....... regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. RESULTS: Using a neoglycoprotein approach, in which a XXXG heptasaccharide...

  3. Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi.

    Science.gov (United States)

    Tanu, Arifur Rahman; Ashraf, Mohammad Arif; Hossain, Md Faruk; Ismail, Md; Shekhar, Hossain Uddin

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development.

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

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

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

  5. Modulation of T cell function by combination of epitope specific and low dose anticytokine therapy controls autoimmune arthritis.

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    Sarah T A Roord

    Full Text Available Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by generating and maintaining inflammation, which leads to tissue damage. Current biological therapies target innate immunity, eminently by interfering with single pro-inflammatory cytokine pathways. This approach has shown excellent efficacy in a good proportion of patients with Rheumatoid Arthritis (RA, but is limited by cost and side effects. Adaptive immunity, particularly T cells with a regulatory function, plays a fundamental role in controlling inflammation in physiologic conditions. A growing body of evidence suggests that modulation of T cell function is impaired in autoimmunity. Restoration of such function could be of significant therapeutic value. We have recently demonstrated that epitope-specific therapy can restore modulation of T cell function in RA patients. Here, we tested the hypothesis that a combination of anti-cytokine and epitope-specific immunotherapy may facilitate the control of autoimmune inflammation by generating active T cell regulation. This novel combination of mucosal tolerization to a pathogenic T cell epitope and single low dose anti-TNFalpha was as therapeutically effective as full dose anti-TNFalpha treatment. Analysis of the underlying immunological mechanisms showed induction of T cell immune deviation.

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2014-12-19

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

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

    Directory of Open Access Journals (Sweden)

    Moustafa Hamze

    2017-05-01

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

  9. The novel carbohydrate epitope L3 is shared by some neural cell adhesion molecules.

    Science.gov (United States)

    Kücherer, A; Faissner, A; Schachner, M

    1987-06-01

    The monoclonal L3 antibody reacts with an N-glycosidically linked carbohydrate structure on at least nine glycoproteins of adult mouse brain. Three out of the L3 epitope-carrying glycoproteins could be identified as the neural cell adhesion molecules L1 and myelin-associated glycoprotein, and the novel adhesion molecule on glia. Expression of the L3 carbohydrate epitope is regulated independently of the protein backbone of these three glycoproteins. Based on the observation that out of three functionally characterized L3 epitope-carrying glycoproteins three fulfill the operational definition of an adhesion molecule, we would like to suggest that they form a new family of adhesion molecules that is distinct from the L2/HNK-1 carbohydrate epitope family of neural cell adhesion molecules. Interestingly, some members in each family appear to be unique to one family while other members belong to the two families.

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

    Directory of Open Access Journals (Sweden)

    Wei Jian-Chao

    2011-07-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  12. 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.

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaohong Li

    2014-01-01

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

  15. Oral immunotherapy for pollen allergy using T-cell epitope-containing egg white derived from genetically manipulated chickens.

    Directory of Open Access Journals (Sweden)

    Yoshinori Kawabe

    Full Text Available 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.

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

    Science.gov (United States)

    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

  17. Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

    Directory of Open Access Journals (Sweden)

    Minglong Liu

    Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Véronique Schulten

    2018-02-01

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

  1. Broadening the repertoire of melanoma-associated T-cell epitopes

    DEFF Research Database (Denmark)

    Frøsig, Thomas Mørch; Lyngaa, Rikke Birgitte; Met, Özcan

    2015-01-01

    . Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase...... and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer...... in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing...

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

    Science.gov (United States)

    Lemmel, Claudia; Stevanović, Stefan

    2003-03-01

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

  3. Single-epitope DNA vaccination prevents exhaustion and facilitates a broad antiviral CD8+ T cell response during chronic viral infection

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard

    2004-01-01

    of DNA vaccines encoding immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV). We analyzed the spectrum of the CD8+ T cell response and the susceptibility to infection in H-2(b) and H-2(d) mice. Priming for a monospecific, CD8+ T cell response did not render mice susceptible to viral...... acute LCMV infection, DNA vaccination did not significantly impair naturally induced immunity. Thus, the response to the other immunogenic epitopes was not dramatically suppressed in DNA-immunized mice undergoing normal immunizing infection, and the majority of mice were protected against rechallenge...... variants. Thus, vaccinated mice were protected against chronic infection with LCMV, and no evidence indicating biologically relevant viral escape was obtained. In parallel, a broad and sustained CD8+ T cell response was generated upon infection, and in H-2(d) mice epitope spreading was observed. Even after...

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

    Science.gov (United States)

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

    2004-12-01

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

  5. Delineation of Several DR-Restricted Tetanus Toxin T Cell Epitopes

    NARCIS (Netherlands)

    Demotz, Stephane; Lanzavecchia, Antonio; Eisel, Ulrich; Niemann, Heiner; Widmann, Christian; Corradin, Giampietro

    1989-01-01

    We have characterized five human T cell clones specific for tetanus toxin. The combination of different techniques allowed us to precisely map two T cell epitopes within fragments 830-843 and 1273-1284 of tetanus toxin, as formally demonstrated by the use of corresponding synthetic peptides. The

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

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

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

  7. Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ruicheng Wei

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

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

    Directory of Open Access Journals (Sweden)

    Wei Zheng

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

  10. Identification and translational validation of novel mammaglobin-A CD8 T cell epitopes

    OpenAIRE

    Soysal, S. D.; Muenst, S.; Kan-Mitchell, J.; Huarte, E.; Zhang, X.; Wilkinson-Ryan, I.; Fleming, T.; Tiriveedhi, V.; Mohanakumar, T.; Li, L.; Herndon, J.; Oertli, D.; Goedegebuure, S. P.; Gillanders, W. E.

    2014-01-01

    Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80 % of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for a breast cancer prevention vaccine, and we recently initiated a phase 1 clinical trial of a MAM-A DNA vaccine. Previously, we have identified multiple MAM-A CD8 T cell epitopes using a reverse immunology candidate epitope approach based on predicted binding, but to date n...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools. (c) 2007 John Wiley & Sons, Ltd....

  12. Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.

    2001-01-01

    We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11...... high antibody titers against the ORF4 ES, In some animals, sera diluted 1:62,500 still gave weak positive enzyme immunoassay reactivity against the ORF4 ES, This hitherto unrecognized immunodominance likely caused phages displaying the ORF4 ES to outcompete phages displaying other ES during library......-term viremic pigs towards some ES, The implications of these findings for PRRSV diagnostics and immunopathogenesis are discussed....

  13. Exposure and binding of selected immunodominant La/SSB epitopes on human apoptotic cells.

    Science.gov (United States)

    Neufing, Petra J; Clancy, Robert M; Jackson, Michael W; Tran, Hai Bac; Buyon, Jill P; Gordon, Tom P

    2005-12-01

    Opsonization of apoptotic cells by autoantibodies bound to surface membrane-translocated La/SSB antigens may initiate tissue damage in the setting of congenital heart block. By injecting pregnant mice with human anti-La antibodies, we previously demonstrated the formation of IgG-apoptotic cell complexes in the developing mouse fetus; however, the binding of anti-La antibodies to human-specific epitopes could not be addressed. Accordingly, the objective of the current study was to delineate the epitope specificity of human La antibodies that are exposed on the surface of apoptotic cells. We used fluorescence microscopy and flow cytometry to assess the binding of human anti-La antibodies affinity purified against immunodominant epitopes of La to human cells undergoing spontaneous apoptosis, in a murine xenograft model in vivo and in cultured human fetal cardiocytes rendered apoptotic in vitro, respectively. Anti-La antibodies bound to immunodominant epitopes of La within the NH(2)-terminus and the RNA recognition motif (RRM) region of apoptotic human cells, in both xenografts and fetal cardiocytes. In contrast, human antibodies affinity purified against the COOH-terminal La epitope did not bind apoptotic cells in either model. This defines the topology of redistributed La during apoptosis, with surface exposure of the NH(2)-terminus and RRM regions. The potential importance of anti-La NH(2)-terminal and anti-La RRM specificity was confirmed by detection of this reactivity in mothers of children with congenital heart block. These findings provide insight into both the molecular modification of the La autoantigen during apoptosis and the specificity of antibodies capable of binding to surface-exposed La. Subsequent formation of surface immune complexes may lead to tissue injury in patients with autoimmune diseases such as congenital heart block.

  14. Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes.

    Science.gov (United States)

    Gangwar, Roopesh Singh; Shil, Pratip; Sapkal, Gajanan N; Khan, Siraj A; Gore, Milind M

    2012-01-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-11-02

    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 2 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 using 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 4 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 4 serotypes as either a prophylactic or therapeutic vaccine.

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

    Science.gov (United States)

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-12-01

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

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

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    Qin Yong-Li

    2011-07-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

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

    2017-07-01

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

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

    Science.gov (United States)

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

  1. Epitope-Specific Vaccination Limits Clonal Expansion of Heterologous Naive T Cells during Viral Challenge

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    Lexus R. Johnson

    2016-10-01

    Full Text Available Despite robust secondary T cell expansion primed by vaccination, the impact on primary immune responses to heterotypic antigens remains undefined. Here we show that secondary expansion of epitope-specific memory CD8+ T cells primed by prior infection with recombinant pathogens limits the primary expansion of naive CD8+ T cells with specificity to new heterologous antigens, dampening protective immunity against subsequent pathogen challenge. The degree of naive T cell repression directly paralleled the magnitude of the recall response. Suppressed primary T cell priming reflects competition for antigen accessibility, since clonal expansion was not inhibited if the primary and secondary epitopes were expressed on different dendritic cells. Interestingly, robust recall responses did not impact antigen-specific NK cells, suggesting that adaptive and innate lymphocyte responses possess different activation requirements or occur in distinct anatomical locations. These findings have important implications in pathogen vaccination strategies that depend on the targeting of multiple T cell epitopes.

  2. Identification of T-cell epitopes using ELISpot and peptide pool arrays.

    Science.gov (United States)

    Tobery, Timothy W; Caulfield, Michael J

    2004-01-01

    Here we describe a method for T-cell epitope identification using a modified ELISpot assay that is both simple and efficient. By using a carefully constructed array of pools of overlapping peptides spanning the entire antigen sequence to stimulate T-cell responses, we are able to detect antigen-specific cytokine responses by both CD8+ and CD4+ T cells and identify the specific peptides to which the cells are responding. Additionally, by performing magnetic bead depletion of either CD8+ or CD4+ cells prior to the assay, we are able to determine the phenotype of the responding cells to each of the peptide epitopes identified. Use of this method will allow the identification of both CD4+ and CD8+ T-cell epitopes without the need for MHC allele-matched reagents and without the need for highly specialized instrumentation. By using an array of peptide pools, this method also dramatically reduces the number of immune cells required to test the entire antigen sequence, often a limiting factor in vaccine testing and other studies.

  3. Prediction of conformational B-cell epitopes from 3D structures by random forests with a distance-based feature

    Directory of Open Access Journals (Sweden)

    Zou Hua

    2011-08-01

    Full Text Available Abstract Background Antigen-antibody interactions are key events in immune system, which provide important clues to the immune processes and responses. In Antigen-antibody interactions, the specific sites on the antigens that are directly bound by the B-cell produced antibodies are well known as B-cell epitopes. The identification of epitopes is a hot topic in bioinformatics because of their potential use in the epitope-based drug design. Although most B-cell epitopes are discontinuous (or conformational, insufficient effort has been put into the conformational epitope prediction, and the performance of existing methods is far from satisfaction. Results In order to develop the high-accuracy model, we focus on some possible aspects concerning the prediction performance, including the impact of interior residues, different contributions of adjacent residues, and the imbalanced data which contain much more non-epitope residues than epitope residues. In order to address above issues, we take following strategies. Firstly, a concept of 'thick surface patch' instead of 'surface patch' is introduced to describe the local spatial context of each surface residue, which considers the impact of interior residue. The comparison between the thick surface patch and the surface patch shows that interior residues contribute to the recognition of epitopes. Secondly, statistical significance of the distance distribution difference between non-epitope patches and epitope patches is observed, thus an adjacent residue distance feature is presented, which reflects the unequal contributions of adjacent residues to the location of binding sites. Thirdly, a bootstrapping and voting procedure is adopted to deal with the imbalanced dataset. Based on the above ideas, we propose a new method to identify the B-cell conformational epitopes from 3D structures by combining conventional features and the proposed feature, and the random forest (RF algorithm is used as the

  4. Differential virus-specific CD8+T-cell epitope repertoire in hepatitis C virus genotype 1 versus 4.

    Science.gov (United States)

    Luxenburger, Hendrik; Graß, Franziska; Baermann, Janina; Boettler, Tobias; Marget, Matthias; Emmerich, Florian; Panning, Marcus; Thimme, Robert; Nitschke, Katja; Neumann-Haefelin, Christoph

    2018-02-03

    Virus-specific CD8 + T-cell responses play an important role in the outcome of hepatitis C virus (HCV) infection. To date, most HCV-specific CD8 + T-cell epitopes have been defined in HCV genotype 1 infection. In contrast, the HCV genotype 4-specific CD8 + T-cell response is poorly defined. Here, we analysed whether known HCV-specific CD8 + T-cell epitopes are also recognized in HCV genotype 4-infected patients and set out to identify the first HCV genotype 4-specific CD8 + T-cell epitopes. We studied patients chronically infected with HCV genotype 1 (n = 20) or 4 (n = 21) using 91 well-described HCV-specific epitope peptides. In addition, we analysed 24 genotype 4-infected patients using 40 epitope candidates predicted using an in silico approach. HCV-specific CD8 + T-cell responses targeting previously described epitopes were detectable in the majority of genotype 1-infected patients (11 of 20). In contrast, patients infected with HCV genotype 4 rarely targeted these epitopes (4 of 21; P = .0247). Importantly, we were able to identify eight novel HCV genotype 4-specific CD8 + T-cell epitopes. Only one of these epitopes was shared between genotype 1 and genotype 4. These results indicate that there is little overlap between CD8 + T-cell repertoires targeting HCV genotype 1 and 4. Prophylactic vaccination studies based on HCV genotype 1 are currently underway. However, in countries with the highest prevalence of HCV infection, such as Egypt, most patients are infected with HCV genotype 4. Thus, prophylactic vaccination strategies need to be adapted to HCV genotype 4 before their application to regions where HCV genotype 4 is endemic. © 2018 John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    Carlos A Parra-López

    Full Text Available 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.

  6. Characterization of Non-Conserved HLA-A*0201 Binding T cell Epitopes of JC Virus T Antigen

    Directory of Open Access Journals (Sweden)

    Jongming Li

    2008-01-01

    Full Text Available JC virus-specific CD8+ cytotoxic T lymphocytes are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy. However, very few JC virus T cell epitopes restricted to MHC class I have been defined. Of the two HLA-A*0201-restricted JCV epitopes, VP1p36 and VP1p100, studies have shown that they are conserved T cell epitopes of polyomaviruses. The cross-recognition associated to these epitopes has complicated the efforts of understanding the dynamics of immune response to JC virus. Based on the previously identified HLA-A*0201 binding T cell epitope of Simian virus 40 T antigen P281–289 (KCDDVLLLL and BK virus T antigen P558–566 (SLQNSEFLL, T cell epitopes of JC Virus T antigen P282–290 (KCEDVFLLM and P557–565 (SLSCSEYLL were identified. In this report, we demonstrated that JC Virus P282–290 P557–565 were able to stimulate T cell responses in healthy donors’ PBMCs and CD8+ cytotoxic T lymphocytes raised with both peptides could recognize and lyse their targets. Most importantly, there were no T cell cross-recognitions between JC Virus, BK Virus and SV40 virus. Therefore, JCV T-ag epitopes P282–290 and P557–565 could be better antigen epitopes compared to VP1p36 and VP1p100 to study the dynamics of cellular immune response to JCV in PML patients. In addition, as a HLA-A*0201 binding T cell epitope, both peptides could be a valuable component of immunotherapies aiming at increasing the cellular immune response against JCV for the treatment of progressive multifocal leukoencephalopathy.

  7. Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells

    NARCIS (Netherlands)

    van den Berg, T. K.; Hasbold, J.; Renardel de Lavalette, C.; Döpp, E. A.; Dijkstra, C. D.; Klaus, G. G.

    1996-01-01

    In this study we describe the tissue distribution of mouse CD40 using two monoclonal antibodies (mAb) against different epitopes of the molecule. In lymphoid tissues CD40 was expressed by B lymphocytes. Most B cells in typical B-cell compartments were CD40-positive, including germinal centre B

  8. Identification of CD8(+) T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Lelic, A.; Parsons, R.

    2010-01-01

    bioinformatics methods to predict WNV-specific CD8(+) T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8(+) T cell...... identified CD8(+) T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8(+) T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine....

  9. CD4+ T-cell activation for immunotherapy of malignancies using Ii-Key/MHC class II epitope hybrid vaccines.

    Science.gov (United States)

    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.

  10. Protective efficacy of serially up-ranked subdominant CD8+ T cell epitopes against virus challenges.

    Directory of Open Access Journals (Sweden)

    Eung-Jun Im

    2011-05-01

    Full Text Available Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Several mechanisms exist to avoid or suppress inflammatory T-cell immune responses that could prove harmful to the host due to targeting self-antigens or commensal microbes. We hypothesized that these mechanisms could become evident when comparing the immunogenicity of a peptide from a pathogen...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially...... the polarization) of T-cell responses to a given epitope is influenced and to some extent predictable based on its similarity to self-antigens and commensal antigens....

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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    Shayla K Shorter

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

  14. T Cell Epitope Immunotherapy Induces a CD4+ T Cell Population with Regulatory Activity

    Directory of Open Access Journals (Sweden)

    Verhoef Adrienne

    2005-01-01

    Full Text Available Background Synthetic peptides, representing CD4+ T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils. Methods and Findings In this study we address the mechanism of action of peptide immunotherapy (PIT in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4+ cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4neg PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4+ and CD8+ PBMCs. Conclusion This study provides evidence for the induction of a population of CD4+ T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

  15. Specific immunotherapy modifies allergen-specific CD4+ T cell responses in an epitope-dependent manner

    Science.gov (United States)

    Wambre, Erik; DeLong, Jonathan H.; James, Eddie A.; Torres-Chinn, Nadia; Pfützner, Wolfgang; Möbs, Christian; Durham, Stephen R.; Till, Stephen J.; Robinson, David; Kwok, William W.

    2014-01-01

    Background Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T cell epitope level is critical for the design of new allergy vaccine strategies. Objective To characterize allergen-specific T cell responses linked with allergy or peripheral tolerance and to determine how CD4+ T cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy (ASIT). Methods Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy and phenotype of the DR04:01-restricted TGP-specific T cell responses in ten grass pollen allergic, five non-atopic and six allergy vaccine-treated individuals was determined using an ex vivo pMHCII-tetramer approach. Results CD4+ T cells in allergic individuals are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. ASIT was associated with preferential deletion of allergen-specific TH2 cells and without significant change in frequency of TH1/TR1 cells. Conclusions Preferential allergen-specific TH2-cells deletion after repeated high doses antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy. PMID:24373351

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

    Science.gov (United States)

    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. Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1.

    Science.gov (United States)

    Etto, Tamara; de Boer, Carmela; Prickett, Sara; Gardner, Leanne M; Voskamp, Astrid; Davies, Janet M; O'Hehir, Robyn E; Rolland, Jennifer M

    2012-01-01

    Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens. Copyright © 2012 S. Karger AG, Basel.

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

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Hoof, Ilka; Lund, Ole

    2010-01-01

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

  19. Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

    Directory of Open Access Journals (Sweden)

    Felix K M Lorenz

    Full Text Available Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

  20. The cancer exome generated by alternative mRNA splicing dilutes predicted HLA class I epitope density.

    Science.gov (United States)

    Stranzl, Thomas; Larsen, Mette V; Lund, Ole; Nielsen, Morten; Brunak, Søren

    2012-01-01

    Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I supertype representatives consistently found to contain fewer predicted epitopes compared to normal tissue. We observed a significant difference in amino acid composition between protein sequences associated with normal versus cancer tissue, as transcripts found in cancer are enriched with hydrophilic amino acids. This variation contributes to the observed significant lower likelihood of cancer-specific peptides to be predicted epitopes compared to peptides found in normal tissue.

  1. The cancer exome generated by alternative mRNA splicing dilutes predicted HLA class I epitope density.

    Directory of Open Access Journals (Sweden)

    Thomas Stranzl

    Full Text Available Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the cell surface. Here, we use a peptidome wide analysis of predicted alternative splice forms, based on a publicly available database, to show that peptides over-represented in cancer splice variants comprise significantly fewer predicted HLA class I epitopes compared to peptides from normal transcripts. Peptides over-represented in cancer transcripts are in the case of the three most common HLA class I supertype representatives consistently found to contain fewer predicted epitopes compared to normal tissue. We observed a significant difference in amino acid composition between protein sequences associated with normal versus cancer tissue, as transcripts found in cancer are enriched with hydrophilic amino acids. This variation contributes to the observed significant lower likelihood of cancer-specific peptides to be predicted epitopes compared to peptides found in normal tissue.

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

    OpenAIRE

    Lundegaard, Claus; Hoof, Ilka; Lund, Ole; Nielsen, Morten

    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. Moreov...

  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

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E

    2014-01-01

    of differentiation on HIV-1-specific CD8+ T-cell populations(n = 128) spanning 11 different epitope targets. RESULTS: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR...

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

    Science.gov (United States)

    Dervillez, Xavier; Gottimukkala, Chetan; Kabbara, Khaled W.; Nguyen, Chelsea; Badakhshan, Tina; Kim, Sarah M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2012-01-01

    Summary Considering the limited success of the recent herpes clinical vaccine trial [1], new vaccine strategies are needed. Infections with herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) in the majority of men and women are usually asymptomatic and results in lifelong viral latency in neurons of sensory ganglia (SG). However, in a minority of men and women HSV spontaneous reactivation can cause recurrent disease (i.e., symptomatic individuals). Our recent findings show that T cells from symptomatic and asymptomatic men and women (i.e. those with and without recurrences, respectively) recognize different herpes epitopes. This finding breaks new ground and opens new doors to assess a new vaccine strategy: mucosal immunization with HSV-1 & HSV-2 epitopes that induce strong in vitro CD4 and CD8 T cell responses from PBMC derived from asymptomatic men and women (designated here as “asymptomatic” protective epitopes”) could boost local and systemic “natural” protective immunity, induced by wild-type infection. Here we highlight the rationale and the future of our emerging “asymptomatic” T cell epitope-based mucosal vaccine strategy to decrease recurrent herpetic disease. PMID:22701511

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

    Directory of Open Access Journals (Sweden)

    Paul Thiamjoo Tan

    2010-01-01

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

  6. An approach to the identification of T cell epitopes in the genomic era: application to Francisella tularensis.

    Science.gov (United States)

    Valentino, Michael; Frelinger, John

    2009-12-01

    The identification and characterization of epitopes is essential for modern immunologic studies. Here, we describe a novel methodology we have developed to identify T cell epitopes exploiting the phenomenon of cross presentation. Particulate antigens, in the form of beads, are very effective in delivering exogenous antigen to both the class I and class II pathways. We will review our efforts to screen entire genomes of pathogens for T cell epitopes taking advantage of the advances in genomics using Francisella tularensis as a model. By automating aspects of this technology we will be able to functionally screen the entire genome of F. tularensis for T cell epitopes. This technology should be applicable not only to F. tularensis, but also to many other pathogens as well.

  7. Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells

    NARCIS (Netherlands)

    G.M.G.M. Verjans (George); C.M. Janssen (Riny); F.G.C.M. Uytdehaag (Fons); C.E.M. van Doornik (C. E M); J. Tommassen (Jan)

    1995-01-01

    textabstractVaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by

  8. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules...

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

    Science.gov (United States)

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

    2015-07-01

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

  10. A Minimum Epitope Overlap between Infections Strongly Narrows the Emerging T Cell Repertoire

    Directory of Open Access Journals (Sweden)

    Susanne G. Oberle

    2016-10-01

    Full Text Available Many infections are caused by pathogens that are similar, but not identical, to previously encountered viruses, bacteria, or vaccines. In such re-infections, pathogens introduce known antigens, which are recognized by memory T cells and new antigens that activate naive T cells. How preexisting memory T cells impact the repertoire of T cells responding to new antigens is still largely unknown. We demonstrate that even a minimum epitope overlap between infections strongly increases the activation threshold and narrows the diversity of T cells recruited in response to new antigens. Thus, minimal cross-reactivity between infections can significantly impact the outcome of a subsequent immune response. Interestingly, we found that non-transferrable memory T cells are most effective in raising the activation threshold. Our findings have implications for designing vaccines and suggest that vaccines meant to target low-affinity T cells are less effective when they contain a strong CD8 T cell epitope that has previously been encountered.

  11. The Challenges and Opportunities for Development of a T-Cell Epitope-Based Herpes Simplex Vaccine

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  14. MHC molecules protect T cell epitopes against proteolytic destruction

    DEFF Research Database (Denmark)

    Mouritsen, S; Meldal, M; Werdelin, O

    1992-01-01

    There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...... that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC...

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

    Science.gov (United States)

    Yu, Tian-Fei; Ma, Bo; Wang, Jun-Wei

    2016-10-15

    Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. Copyright © 2016. Published by Elsevier B.V.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  18. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus

    2011-01-01

    bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized......Background: CD4+ T cells orchestrate immune protection by ‘‘helping’’ other cells of our immune system to clear viral infections. It is well known that the preferential infection and depletion of CD4+ T cells contributes to hampered systemic T cell help following HIV infection. However......, the functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...

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

    DEFF Research Database (Denmark)

    Adorini, L; Sette, A; Buus, S

    1988-01-01

    The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis...... of the T-cell proliferative response induced by synthetic peptides covering almost the entire HEL sequence. All the T-cell hybridomas from H-2d mice analyzed recognize the HEL sequence 107-116 in association with the I-Ed molecule. Correlating with the restriction of T-cell recognition, HEL-(105......-120)-peptide binds to I-Ed but not to I-Ad molecules. Conservative or semiconservative substitutions at positions 113 (Asn----Lys), 114 (Arg----His), or 115 (Cys----Ala) abrogate the ability of HEL-(105-120) to activate T cells. Substitutions at residues 113 and 115 affect T-cell recognition...

  20. Identification of Protective B-Cell Epitopes within the Novel Malaria Vaccine Candidate Plasmodium falciparum Schizont Egress Antigen 1.

    Science.gov (United States)

    Nixon, Christina E; Park, Sangshin; Pond-Tor, Sunthorn; Raj, Dipak; Lambert, Lynn E; Orr-Gonzalez, Sachy; Barnafo, Emma K; Rausch, Kelly M; Friedman, Jennifer F; Fried, Michal; Duffy, Patrick E; Kurtis, Jonathan D

    2017-07-01

    Naturally acquired antibodies to Plasmodium falciparum schizont egress antigen 1 (PfSEA-1A) are associated with protection against severe malaria in children. Vaccination of mice with SEA-1A from Plasmodium berghei (PbSEA-1A) decreases parasitemia and prolongs survival following P. berghei ANKA challenge. To enhance the immunogenicity of PfSEA-1A, we identified five linear B-cell epitopes using peptide microarrays probed with antisera from nonhuman primates vaccinated with recombinant PfSEA-1A (rPfSEA-1A). We evaluated the relationship between epitope-specific antibody levels and protection from parasitemia in a longitudinal treatment-reinfection cohort in western Kenya. Antibodies to three epitopes were associated with 16 to 17% decreased parasitemia over an 18-week high transmission season. We are currently designing immunogens to enhance antibody responses to these three epitopes. Copyright © 2017 American Society for Microbiology.

  1. Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

    Science.gov (United States)

    Solache, A; Morgan, C L; Dodi, A I; Morte, C; Scott, I; Baboonian, C; Zal, B; Goldman, J; Grundy, J E; Madrigal, J A

    1999-11-15

    The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.

  2. Variable epitope library-based vaccines: shooting moving targets.

    Science.gov (United States)

    Pedroza-Roldan, Cesar; Charles-Niño, Claudia; Saavedra, Rafael; Govezensky, Tzipe; Vaca, Luis; Avaniss-Aghajani, Eric; Gevorkian, Goar; Manoutcharian, Karen

    2009-12-01

    While the antigenic variability is the major obstacle for developing vaccines against antigenically variable pathogens (AVPs) and cancer, this issue is not addressed adequately in current vaccine efforts. We developed a novel variable epitope library (VEL)-based vaccine strategy using immunogens carrying a mixture of thousands of variants of a single epitope. In this proof-of-concept study, we used an immunodominant HIV-1-derived CD8+ cytotoxic T-lymphocyte (CTL) epitope as a model antigen to construct immunogens in the form of plasmid DNA and recombinant M13 bacteriophages. We generated combinatorial libraries expressing epitope variants with random amino acid substitutions at 2-5 amino acid positions within the epitope. Mice immunized with these immunogens developed epitope-specific CD8+ IFN-gamma+ T-cell responses that recognized more than 50% of heavily mutated variants of wild-type epitope, as demonstrated in T-cell proliferation assays and FACS analysis. Strikingly, these potent and broad epitope-specific immune responses were long lasting: after 12 months of priming, epitope variants were recognized by CD8+ cells and effector memory T cells were induced. In addition, we showed, for the first time, the inhibition of T-cell responses at the molecular level by immune interference: the mice primed with wild-type epitope and 8 or 12 months later immunized with VELs, were not able to recognize variant epitopes efficiently. These data may give a mechanistic explanation for the failure of recent HIV vaccine trials as well as highlight specific hurdles in current molecular vaccine efforts targeting other important antigenically variable pathogens and diseases. These findings suggest that the VEL-based strategy for immunogen construction can be used as a reliable technological platform for the generation of vaccines against AVPs and cancer, and contribute to better understanding complex host-pathogen interactions.

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

    Science.gov (United States)

    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.

  4. Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes

    DEFF Research Database (Denmark)

    Kloverpris, Henrik; Karlsson, Ingrid; Bonde, Jesper

    2009-01-01

    with seven CD8 T-cell epitopes and three CD4 T-cell epitopes. Epitope-specific responses were evaluated by intracellular cytokine staining for interferon-gamma, tumor necrosis factor alpha and interleukin-2 and/or pentamer labeling 3 weeks prior to, 10 weeks after and 32 weeks after the first immunization....... RESULTS:: Previously undetected T-cell responses specific for one or more epitopes were induced in all 12 individuals. Half of the participants had sustained CD4 T-cell responses 32 weeks after immunization. No severe adverse effects were observed. No overall or sustained change in viral load or CD4 T...

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Saghar Kaabinejadian

    Full Text Available 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.

  7. Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project."

    DEFF Research Database (Denmark)

    Lauemøller, S L; Kesmir, C; Corbet, S L

    2000-01-01

    discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles...... processing, as these become available. The ability to translate the accumulating primary sequence databases in terms of immune recognition should enable scientists and clinicians to analyze any protein of interest for the presence of potentially immunogenic epitopes. The computational tools to scan entire...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    Science.gov (United States)

    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. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    DEFF Research Database (Denmark)

    Tang, Sheila Tuyet; Wang, M.; Lamberth, K.

    2008-01-01

    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......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...

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

    Directory of Open Access Journals (Sweden)

    Hitchen Paul G

    2011-03-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions 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.

  15. Identification of helper T cell epitopes of dengue virus E-protein.

    Science.gov (United States)

    Leclerc, C; Dériaud, E; Megret, F; Briand, J P; Van Regenmortel, M H; Deubel, V

    1993-05-01

    The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides. Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice. Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E. coli as polypeptides fused to trpE (f22-205, f267-354, f366-424). To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes. Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359. Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes. Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice. This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design.

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

    Directory of Open Access Journals (Sweden)

    Daniel J Hui

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

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

    Directory of Open Access Journals (Sweden)

    Stefanie Ameres

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

  18. Mechanisms, safety and efficacy of a B cell epitope-based vaccine for immunotherapy of grass pollen allergy

    Directory of Open Access Journals (Sweden)

    Petra Zieglmayer

    2016-09-01

    Conclusion: The B cell epitope-based recombinant grass pollen allergy vaccine BM32 is well tolerated and few doses are sufficient to suppress immediate allergic reactions as well as allergen-specific T cell responses via a selective induction of allergen-specific IgG antibodies. (ClinicalTrials.gov number, NCT01445002.

  19. In silico CD4+, CD8+ T-cell and B-cell immunity associated immunogenic epitope prediction and HLA distribution analysis of Zika virus.

    Science.gov (United States)

    Janahi, Essam Mohammed; Dhasmana, Anupam; Srivastava, Vandana; Sarangi, Aditya Narayan; Raza, Sana; Arif, Jamal M; Bhatt, Madan Lal Bramha; Lohani, Mohtashim; Areeshi, Mohammed Yahya; Saxena, Anand Murari; Haque, Shafiul

    2017-01-01

    Zika virus (ZIKV) is a mosquito-borne flavivirus distributed all over Africa, South America and Asia. The infection with the virus may cause acute febrile sickness that clinically resembles dengue fever, yet there is no vaccine, no satisfactory treatment, and no means of evaluating the risk of the disease or prognosis in the infected people. In the present study, the efficacy of the host's immune response in reducing the risk of infectious diseases was taken into account to carry out immuno-informatics driven epitope screening strategy of vaccine candidates against ZIKV. In this study, HLA distribution analysis was done to ensure the coverage of the vast majority of the population. Systematic screening of effective dominant immunogens was done with the help of Immune Epitope & ABCPred databases. The outcomes suggested that the predicted epitopes may be protective immunogens with highly conserved sequences and bear potential to induce both protective neutralizing antibodies, T & B cell responses. A total of 25 CD4+ and 16 CD8+ peptides were screened for T-cell mediated immunity. The predicted epitope "TGLDFSDLYYLTMNNKHWLV" was selected as a highly immunogenic epitope for humoral immunity. These peptides were further screened as non-toxic, immunogenic and non-mutated residues of envelop viral protein. The predicted epitope could work as suitable candidate(s) for peptide based vaccine development. Further, experimental validation of these epitopes is warranted to ensure the potential of B- and T-cells stimulation for their efficient use as vaccine candidates, and as diagnostic agents against ZIKV.

  20. B-cell epitopes in GroEL of Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Zhaohua Lu

    Full Text Available 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.

  1. Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes

    Science.gov (United States)

    Volcovich, Romina; Altcheh, Jaime; Bracamonte, Estefanía; Marco, Jorge D.; Nielsen, Morten; Buscaglia, Carlos A.

    2017-01-01

    Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas’ disease diagnosis. PMID:28991925

  2. Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes.

    Directory of Open Access Journals (Sweden)

    Juan Mucci

    2017-10-01

    Full Text Available Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects. The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas' disease diagnosis.

  3. Identification of Candidate Tolerogenic CD8+ T Cell Epitopes for Therapy of Type 1 Diabetes in the NOD Mouse Model

    Directory of Open Access Journals (Sweden)

    Cailin Yu

    2016-01-01

    Full Text Available Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet β cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8+ T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8+ T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158–166 and 282–290 and one in a non-β cell protein, dopamine β-hydroxylase (aa 233–241. Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DβH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DβH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

  4. 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

    DEFF Research Database (Denmark)

    Steffensen, Maria A; Pedersen, Louise Holm; Jahn, Marie Louise

    2016-01-01

    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......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...

  5. Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope.

    Science.gov (United States)

    Wen, Xiaobo; Wei, Xiaoman; Ran, Xuhua; Ni, Hongbo; Cao, Si; Zhang, Yao

    2015-08-26

    Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64-223) recombinant protein with or without a universal tetanus toxoid CD4(+) T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10 μg/dose) or guinea pigs (20 μg/dose) immunized IM with 600 μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Neo-Epitopes Generated on Hydroxyl Radical Modified GlycatedIgG Have Role in Immunopathology of Diabetes Type 2.

    Directory of Open Access Journals (Sweden)

    Sidra Islam

    Full Text Available Glycoxidation plays a crucial role in diabetes and its associated complications. Among the glycoxidation agents, methylglyoxal (MG is known to have very highglycationpotential witha concomitant generation of reactive oxygen species (ROS during its synthesis and degradation. The presentstudy probes the MG and ROSinduced structural damage to immunoglobulin G (IgG and alterations in its immunogenicity in diabetes type 2 patients (T2DM. Human IgG was first glycated with MG followed by hydroxyl radical (OH• modification. Glycoxidation mediated effects on IgG were evaluated by various physicochemical techniques likeultraviolet (UV and fluorescence spectroscopy, 8-anilinonaphthalene-1-sulfonic acid (ANS binding studies, carbonyl andfree sulfhydryl groups assay, matrix assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF, red blood cell (RBC haemolysis assay, Congored (CR staining analysis and scanning electron microscopy (SEM. The results revealed hyperchromicityin UV, advanced glycation end product (AGEspecific and ANS fluorescence, quenching in tyrosine and tryptophan fluorescence intensity,enhanced carbonyl content,reduction in free sulfhydryl groups,pronounced shift in m/z value of IgGand decrease in antioxidant activity in RBC induced haemolysis assayupon glycoxidation. SEM and CRstaining assay showed highly altered surface morphology in glycoxidised sample as compared to the native. Enzyme linked immunosorbent assay (ELISA and band shift assay were performed to assess the changes in immunogenicity of IgG upon glyoxidation and its role in T2DM. The serum antibodies derived from T2DM patients demonstrated strong affinity towards OH• treated MG glycatedIgG (OH•-MG-IgG when compared to native IgG (N-IgG or IgGs treated with MG alone (MG-IgG or OH• alone (OH•-IgG. This study shows the cumulating effect of OH• on the glycation potential of MG. The results point towards the modification of IgG in diabetes patients

  7. Compare the Difference of B-cell Epitopes of EgAgB1 and EgAgB3 Proteins Selected through Bioinformatic Analysis

    Science.gov (United States)

    An, Mengting; Zhang, Fengbo; Zhu, Yuejie; Zhao, Xiao; Ding, Jianbing

    2018-01-01

    Cystic echinococcosis, as a zoonosis, seriously endangers humans and animals, so early diagnosis of this disease is particularly important. Therefore, this study is to predict B-cell epitopes of EgAgB1 and EgAgB3 proteins by bioinformatics software. B-cell epitopes of EgAgB1 and EgAgB3 proteins are predicted using DNAStar and IEDB software. The results suggest that there are two potential B-cell epitopes in EgAgB1, which located in the 8-15 and 31-37 amino acid residue segments. And two potential B-cell epitopes in EgAgB2, located in the 20∼27 and 47∼53 amino acid residue segments. This study predicted the B-cell epitopes of EgAgB1 and EgAgB3 proteins, which laid the foundation for the early diagnosis of Cystic echinococcosis.

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

    Science.gov (United States)

    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.

  9. Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Brock, I; Weldingh, K; Leyten, EM

    2004-01-01

    Specific T-cell epitopes for immunoassay-based diagnosis of Mycobacterium tuberculosis infection.Brock I, Weldingh K, Leyten EM, Arend SM, Ravn P, Andersen P. Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. The currently used...... method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific for Mycobacterium tuberculosis to replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four...... selected and combined the specific peptide stretches from the four proteins not recognized by M. bovis BCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthy M. bovis...

  10. Rapid Antigen Processing and Presentation of a Protective and Immunodominant HLA-B*27-restricted Hepatitis C Virus-specific CD8+ T-cell Epitope

    Science.gov (United States)

    Schmidt, Julia; Iversen, Astrid K. N.; Tenzer, Stefan; Gostick, Emma; Price, David A.; Lohmann, Volker; Distler, Ute; Bowness, Paul; Schild, Hansjörg; Blum, Hubert E.; Klenerman, Paul

    2012-01-01

    HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope. PMID:23209413

  11. A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

    DEFF Research Database (Denmark)

    Würtzen, Peter A; Claesson, Mogens H

    2002-01-01

    , the CTL line, which expressed relatively low affinity for the HLA-A2/peptide complex, was able to kill 3 different HLA-A2(+) p53 mutated tumor cell lines. The present and our previous observations expand the number of p53-derived peptides suitable for vaccination protocols for cancer patients with p53......A p53 peptide-specific CTL line was generated through stimulation with autologous monocyte-derived dendritic cells (DC) pulsed with wild-type HLA-A2 binding p53 derived peptides. A p53 peptide-specific CD8(+) CTL line was established from a healthy HLA-A2 positive donor. The CTL line...... was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA...

  12. A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.

    Directory of Open Access Journals (Sweden)

    Daniela Santoro Rosa

    Full Text Available T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+ T cells are important for the generation and maintenance of functional CD8(+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18, capable of eliciting broad CD4(+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+/CD8(+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+ and CD8(+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2 simultaneously in response to HIV-1 peptides. For CD4(+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2. The vaccine also generated long-lived central and effector memory CD4(+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+ T cells and antibody responses- elicited by other HIV immunogens.

  13. Benchmarking B-Cell Epitope Prediction for the Design of Peptide-Based Vaccines: Problems and Prospects

    Directory of Open Access Journals (Sweden)

    Salvador Eugenio C. Caoili

    2010-01-01

    Full Text Available To better support the design of peptide-based vaccines, refinement of methods to predict B-cell epitopes necessitates meaningful benchmarking against empirical data on the cross-reactivity of polyclonal antipeptide antibodies with proteins, such that the positive data reflect functionally relevant cross-reactivity (which is consistent with antibody-mediated change in protein function and the negative data reflect genuine absence of cross-reactivity (rather than apparent absence of cross-reactivity due to artifactual masking of B-cell epitopes in immunoassays. These data are heterogeneous in view of multiple factors that complicate B-cell epitope prediction, notably physicochemical factors that define key structural differences between immunizing peptides and their cognate proteins (e.g., unmatched electrical charges along the peptide-protein sequence alignments. If the data are partitioned with respect to these factors, iterative parallel benchmarking against the resulting subsets of data provides a basis for systematically identifying and addressing the limitations of methods for B-cell epitope prediction as applied to vaccine design.

  14. In silico analysis of MHC-I restricted epitopes of Chikungunya virus proteins: Implication in understanding anti-CHIKV CD8(+) T cell response and advancement of epitope based immunotherapy for CHIKV infection.

    Science.gov (United States)

    Pratheek, B M; Suryawanshi, Amol R; Chattopadhyay, Soma; Chattopadhyay, Subhasis

    2015-04-01

    Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus, responsible for acute febrile infection. The high morbidity and socio-economic loss associated with the recent CHIKV epidemics worldwide have raised a great public health concern and emphasize the need to study the immunological basis of CHIKV infection to control the disease. MHC-I restricted CD8(+) T cell response represent one of the major anti-viral immune responses. Accordingly, it is essential to have a detailed understanding towards CHIKV specific MHC-I restricted immunogenic epitopes for anti-viral CD8(+) CTL immunogenicity. In the present study, a computational approach was used to predict the conserved MHC-I epitopes for mouse haplotypes (H2-Db and H2-Dd) and some alleles of the major HLA-I supertypes (HLA-A2, -A3, -A24, -B7, -B15) of all CHIKV proteins. Further, an in-depth computational analysis was carried out to validate the selected epitopes for their nature of conservation in different global CHIKV isolates to assess their binding affinities to the appropriate site of respective MHC-I molecules and to predict anti-CHIKV CD8(+) CTL immunogenicity. Our analyses resulted in fifteen highly conserved epitopes for H2-Db and H2-Dd and fifty epitopes for different HLA-I supertypes. Out of these, the MHC-I epitopes VLLPNVHTL and MTPERVTRL were found to have highest predictable CTL immunogenicities and least binding energies for H2-Db and H2-Dd, whereas, for HLA-I, the epitope FLTLFVNTL was with the highest population coverage, CTL immunogenicity and least binding energy. Hence, our study has identified MHC-I restricted epitopes that may help in the advancement of MHC-I restricted epitope based anti-CHIKV immune responses against this infection and this will be useful towards the development of epitope based anti-CHIKV immunotherapy in the future. However, further experimental investigations for cross validation and evaluation are warranted to establish the ability of epitopes to induce CD8(+) T cell

  15. Therapeutic vaccination using cationic liposome-adjuvanted HIV type 1 peptides representing HLA-supertype-restricted subdominant T cell epitopes

    DEFF Research Database (Denmark)

    Román, Victor Raúl Gómez; Jensen, Kristoffer Jarlov; Jensen, Sanne Skov

    2013-01-01

    We have designed a therapeutic HIV-1 vaccine concept based on peptides together with the adjuvant CAF01. Peptides represented 15 HLA-supertype-restricted subdominant and conserved CD8 T cell epitopes and three CD4 T-helper cell epitopes. In this phase I clinical trial, safety and immunogenicity...... were assessed in untreated HIV-1-infected individuals in Guinea-Bissau, West Africa. Twenty-three HIV-1-infected individuals were randomized to receive placebo (n=5) or vaccine (n=18). Safety was appraised by clinical follow-up combined with monitoring of biochemistry, hematology, CD4 T cell counts......, and HIV-1 viral loads. T cell immunogenicity was monitored longitudinally by interferon (IFN)-γ ELISpot. New vaccine-specific T cell responses were induced in 6/14 vaccinees for whom ELISpot data were valid. CD4 T cell counts and viral loads were stable. The study shows that therapeutic immunization...

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

    OpenAIRE

    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 app...

  17. Generation and characterization of recombinant human antibodies specific for native laminin epitopes. Potential application in cancer therapy. Cancer Immunol. Immunother

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Russell, Stephen J.

    2001-01-01

    indicated that these antibodies recognize the native protein through conformational epitopes. All of them reduced tumor cell attachment to laminin, suggesting that domains of the laminin molecule that are recognized by these antibodies likely bind to cell-surface receptors. The antibody fragments bind...... to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications....... of single chain antibodies was selected using intact mouse laminin, and five different clones were identified. All the antibodies were specific for their cognate antigen, as revealed by lack of cross-reactivity with other components of the basement membranes. A more extensive characterization of the panel...

  18. Identification of a protective B-cell epitope of the Staphylococcus aureus GapC protein by screening a phage-displayed random peptide library.

    Directory of Open Access Journals (Sweden)

    Mengyao Wang

    Full Text Available The impact of epidemic Staphylococcus aureus (S. aureus on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of S. aureus is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of S. aureus, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response in vivo. However, the B-cell epitopes of S. aureus GapC have not been well identified. Here we used the bioinformatics tools to analyze the sequence of GapC, and we generated protective anti-GapC monoclonal antibodies (mAbs. A protective mAb (1F4 showed strong specificity to GapC and the ability to induce macrophages to phagocytose S. aureus. We screened the motif 272GYTEDEIVSSD282, which was recognized by mAb 1F4, using a phage display system. Then, we used site-directed mutagenesis to identify key amino acids in the motif. Residues G272 D276 E277 I278 and V279 formed the core of the 272GYTEDEIVSSD282 motif. In addition, we showed that this epitope peptide induced a protective humoral immune response against S. aureus infection in immunized mice. Our results will be useful for the further study of epitope-based vaccines against S. aureus infection.

  19. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Worning, Peder

    2003-01-01

    calculations we show that peptides that bind to the HLA A*0204 complex display signal of higher order sequence correlations. Neural networks are ideally suited to integrate such higher order correlations when predicting the binding affinity. It is this feature combined with the use of several neural networks......In this paper we describe an improved neural network method to predict T-cell class I epitopes. A novel input representation has been developed consisting of a combination of sparse encoding, Blosum encoding, and input derived from hidden Markov models. We demonstrate that the combination...... of several neural networks derived using different sequence-encoding schemes has a performance superior to neural networks derived using a single sequence-encoding scheme. The new method is shown to have a performance that is substantially higher than that of other methods. By use of mutual information...

  20. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  1. Immunoinformatics Approach in Designing Epitope-based Vaccine against Meningitis-inducing Bacteria (, and Type b

    Directory of Open Access Journals (Sweden)

    Hilyatuz Zahroh

    2016-01-01

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

  2. Identification of critical residues of linear B cell epitope on Goodpasture autoantigen.

    Directory of Open Access Journals (Sweden)

    Xiao-yu Jia

    Full Text Available The autoantigen of anti-glomerular basement membrane (GBM disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IVNC1. Our previous study revealed a peptide on α3(IVNC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150. This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA.We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001. P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001 of antibody reaction after each residue replacement.We defined GFxF (α3142, 143,145 as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.

  3. Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Nagesh R Aragam

    Full Text Available Circumsporozoite protein (CS is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates and Malawi (235 isolates, we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

  4. Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions.

    Science.gov (United States)

    Aragam, Nagesh R; Thayer, Kelly M; Nge, Nabi; Hoffman, Irving; Martinson, Francis; Kamwendo, Debbie; Lin, Feng-Chang; Sutherland, Colin; Bailey, Jeffrey A; Juliano, Jonathan J

    2013-01-01

    Circumsporozoite protein (CS) is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates) and Malawi (235 isolates), we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

  5. Immune Epitope Database and Analysis Resource (IEDB)

    Data.gov (United States)

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

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

    Science.gov (United States)

    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. Published by Elsevier Inc.

  7. Circulating Memory CD4+ T Cells Target Conserved Epitopes of Rhinovirus Capsid Proteins and Respond Rapidly to Experimental Infection in Humans.

    Science.gov (United States)

    Muehling, Lyndsey M; Mai, Duy T; Kwok, William W; Heymann, Peter W; Pomés, Anna; Woodfolk, Judith A

    2016-10-15

    Rhinovirus (RV) is a major cause of common cold and an important trigger of acute episodes of chronic lung diseases. Antigenic variation across the numerous RV strains results in frequent infections and a lack of durable cross-protection. Because the nature of human CD4 + T cells that target RV is largely unknown, T cell epitopes of RV capsid proteins were analyzed, and cognate T cells were characterized in healthy subjects and those infected by intranasal challenge. Peptide epitopes of the RV-A16 capsid proteins VP1 and VP2 were identified by peptide/MHC class II tetramer-guided epitope mapping, validated by direct ex vivo enumeration, and interrogated using a variety of in silico methods. Among noninfected subjects, those circulating RV-A16-specific CD4 + T cells detected at the highest frequencies targeted 10 unique epitopes that bound to diverse HLA-DR molecules. T cell epitopes localized to conserved molecular regions of biological significance to the virus were enriched for HLA class I and II binding motifs, and constituted both species-specific (RV-A) and pan-species (RV-A, -B, and -C) varieties. Circulating epitope-specific T cells comprised both memory Th1 and T follicular helper cells, and were rapidly expanded and activated after intranasal challenge with RV-A16. Cross-reactivity was evidenced by identification of a common *0401-restricted epitope for RV-A16 and RV-A39 by tetramer-guided epitope mapping and the ability for RV-A16-specific Th1 cells to proliferate in response to their RV-A39 peptide counterpart. The preferential persistence of high-frequency RV-specific memory Th1 cells that recognize a limited set of conserved epitopes likely arises from iterative priming by previous exposures to different RV strains. Copyright © 2016 by The American Association of Immunologists, Inc.

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Toru Ichihashi

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

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

    Science.gov (United States)

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

    2013-01-01

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

  11. Identification of B cell recognized linear epitopes in a snake venom serine proteinase from the central American bushmaster Lachesis stenophrys.

    Science.gov (United States)

    Madrigal, M; Alape-Girón, A; Barboza-Arguedas, E; Aguilar-Ulloa, W; Flores-Díaz, M

    2017-12-15

    Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein-kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Sterile immunity to malaria after DNA prime/adenovirus boost immunization is associated with effector memory CD8+T cells targeting AMA1 class I epitopes.

    Directory of Open Access Journals (Sweden)

    Martha Sedegah

    Full Text Available Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1 and boosted with human adenovirus-5 (Ad expressing the same antigens (DNA/Ad. Four volunteers (27% demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA did not develop sterile protection.We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the

  14. Elucidating the immunological effects of 5-azacytidine treatment in patients with myelodysplastic syndrome and identifying new conditional ligands and T-cell epitopes of relevance in melanoma

    DEFF Research Database (Denmark)

    Frøsig, Thomas Mørch

    2015-01-01

    This review is focused on research within three different areas of tumor immunology: discovery of new T-cell epitopes and a new immunological antigen (reported in Paper I and II), elucidation of the immunological effects of treatment with a hypomethylating drug (reported in Paper III) and discovery...... of new conditional ligands (reported in Paper IV). Many melanoma-associated T-cell epitopes have been described, but 45% of these are restricted to human leukocyte antigen (HLA)-A2, leaving the remaining 36 different HLA molecules with only a few described T-cell epitopes each. Therefore we wanted...... frequently recognized by T cells from HLA-A2 patients. On contrary, in Paper II we wanted to investigate the protein Nodal as a novel immunological target. We took advantage of a T-cell epitope mapping platform in which HLA ligands are predicted by computer-based algorithms, further tested in the laboratory...

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

    International Nuclear Information System (INIS)

    Yang, Marie; Mine, Yoshinori

    2009-01-01

    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)-γ 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.

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

    Science.gov (United States)

    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.

  17. Induction of immune responses and clinical efficacy in a phase II trial of IDM-2101, a 10-epitope cytotoxic T-lymphocyte vaccine, in metastatic non-small-cell lung cancer.

    Science.gov (United States)

    Barve, Minal; Bender, James; Senzer, Neil; Cunningham, Casey; Greco, F Anthony; McCune, David; Steis, Ronald; Khong, Hung; Richards, Donald; Stephenson, Joe; Ganesa, Prasanthi; Nemunaitis, Jackie; Ishioka, Glenn; Pappen, Beena; Nemunaitis, Michael; Morse, Michael; Mills, Bonnie; Maples, Phillip B; Sherman, Jeffrey; Nemunaitis, John J

    2008-09-20

    Generation of broad cytotoxic T-lymphocyte responses against multiple epitopes and tumor-associated antigens (TAAs) may provide effective immunotherapy in patients with cancer. We evaluated a single-vial peptide vaccine consisting of nine HLA-A2 supertype-binding epitopes (two native and seven analog epitopes modified for optimal HLA binding or T-cell receptor stimulation) covering five TAAs and the universal helper pan-DR epitope, formulated as a stable emulsion with incomplete Freund's adjuvant (Montanide ISA 51; Seppic SA, Paris, France). The clinical efficacy, safety, and multiepitope immunogenicity of IDM-2101 was evaluated in patients with stage IIIB or IV non-small-cell lung cancer (NSCLC). A total of 63 patients were enrolled who were positive for HLA-A2. End points included survival, safety, and immune response. IDM-2101 (previously EP-2101) was administered every 3 weeks for the first 15 weeks, then every 2 months through year 1, then quarterly through year 2, for a total of 13 doses. Epitope-specific cytotoxic and helper T-lymphocyte immunogenic responses were measured by the interferon gamma enzyme-linked immunosorbent spot assay. No significant adverse events were noted. Low-grade erythema and pain at the injection site were the most common adverse effects. One-year survival in the treated patients was 60%, and median survival was 17.3 months. One complete and one partial response were identified. Survival was longer in patients demonstrating an immune response to epitope peptides (P IDM-2101 was well tolerated, and evidence of efficacy was suggested.

  18. Old and New World arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein 2, which is recognized by Lassa virus-specific human CD4+ T-cell clones

    International Nuclear Information System (INIS)

    Meulen, Jan ter; Badusche, Marlis; Satoguina, Judith; Strecker, Thomas; Lenz, Oliver; Loeliger, Cornelius; Sakho, Mohamed; Koulemou, Kekoura; Koivogui, Lamine; Hoerauf, Achim

    2004-01-01

    Data from human studies and animal experiments indicate a dominant role of T-cells over antibodies in controlling acute Lassa virus infection and providing immunity to reinfection. Knowledge of the epitopes recognized by T-cells may therefore be crucial to the development of a recombinant Lassa virus vaccine. In order to study human T-cell reactivity to the most conserved structural protein of Lassa virus, the glycoprotein 2 (GP2), seven GP2-specific CD4+ T-cell clones (TCCs) were generated from the lymphocytes of a Lassa antibody positive individual. All TCC displayed high specific proliferation, showed DR-restriction, and produced IFN-γ upon stimulation with recombinant GP2. The epitope of four of the clones was localized to a short stretch of 13 amino acids located in the N-terminal part of GP2 (aa 289-301, numbering according to sequence of GPC). This epitope is conserved in all strains of Lassa virus and lymphocytic choriomeningitis virus (LCMV), shows >90% similarity in all New World arenaviruses of clade B, and overlaps with the proposed fusion domain of GP2. Peptides with conservative aa exchanges, as they naturally occur in the epitope 289-301 of the Old World arenavirus Mopeia and some New World arenaviruses, continued to effectively stimulate the Lassa-GP2-specific T-cell clones tested. The finding of a human T-helper cell epitope, which is highly conserved between Old and New World arenaviruses, is of importance for the design of arenavirus vaccines

  19. Induction of cell-mediated immunity against mycobacterium tuberculosis using DNA vaccines encoding cytotoxic and helper T-cell epitopes of the 38-kilodalton protein

    NARCIS (Netherlands)

    Fonseca, DPAJ; Benaissa-Trouw, B; Kraaijeveld, CA; Snippe, H; Verheul, AFM

    Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their

  20. Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.

    Science.gov (United States)

    Brossart, P; Heinrich, K S; Stuhler, G; Behnke, L; Reichardt, V L; Stevanovic, S; Muhm, A; Rammensee, H G; Kanz, L; Brugger, W

    1999-06-15

    The tumor-associated antigen MUC1 is overexpressed on various hematological and epithelial malignancies and is therefore a suitable candidate for broadly applicable vaccine therapies. It was demonstrated that major histocompatibility complex (MHC)-unrestricted cytotoxic T cells can recognize epitopes of the MUC1 protein core localized in the tandem repeat domain. There is increasing evidence now that MHC-restricted T cells can also be induced after immunization with the MUC1 protein or segments of the core tandem repeat. Using a computer analysis of the MUC1 amino acid sequence, we identified two novel peptides with a high binding probability to the HLA-A2 molecule. One of the peptides is derived from the tandem repeat region and the other is derived from the leader sequence of the MUC1 protein, suggesting that, in contrast to previous reports, the MUC1-directed immune responses are not limited to the extracellular tandem repeat domain. Cytotoxic T cells (CTL) were generated from several healthy donors by primary in vitro immunization using peptide-pulsed dendritic cells. The addition of a Pan-HLA-DR binding peptide PADRE as a T-helper epitope during the in vitro priming resulted in an increased cytotoxic activity of the MUC1-specific CTL and a higher production of cytokines such as interleukin-12 and interferon-gamma in the cell cultures, demonstrating the importance of CD4 cells for an efficient CTL priming. The peptide induced CTL lysed tumors endogenously expressing MUC1 in an antigen-specific and HLA-A2-restricted fashion, including breast and pancreatic tumor cells as well as renal cell carcinoma cells, showing that these peptides are shared among many tumors. The use of MUC1-derived peptides could provide a broadly applicable approach for the development of dendritic cell-based vaccination therapies.

  1. Analysis of Swine Leukocyte Antigen Peptide Binding Profiles and the Identification of T cell Epitopes by Tetramer Staining

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers

    ) class I molecules, which are highly polymorphic peptide receptors which select and present endogenously derived peptides to circulating CTLs. Peptides that are recognized by CTLs in the context of MHC are epitopes, and represent a small sample of the pathogen proteome, making it possible for the immune...... fingerprint” of an individual can be identified by defining MHC alleles. This is classically called “tissue typing” and is done by analyzing the reactivity of peripheral blood cells with sera unique to MHC alleles. Such knowledge is paramount to analysis of the immune response regarding MHC restriction...... within a species makes immune escape almost impossible for any intruding pathogen. Characterization of the SLA class I and class II gene products and their peptide binding capacity defines the T cell epitopes of any given pathogen proteome. To date the analysis of MHC peptide interactions, strength...

  2. High-throughput discovery of T cell epitopes in type 1 diabetes using DNA barcode labelledpeptide-MHC multimers

    DEFF Research Database (Denmark)

    Lyngaa, Rikke Birgitte; Bentzen, Amalie Kai; Overgaard, A. Julie

    2016-01-01

    applying a novel technology where the selection of MHC-multimer binding T cells is followed by amplification and sequencing of MHC multimer-associated DNA barcodes revealing their recognition. This technique enables simultaneous detection of >1000 specificities. Identifying post translational modifications...... as T cell targets in other autoimmune diseases. We used netMHC prediction algorithm to identify 764 epitopes from Insulin, GAD65, IA-2 and ZnT8 restricted to HLA-A2, A24, B8 and B15. Among these 91 peptide sequences were susceptible for citrullination. We evaluate the MHC-affinity of both...... the citrullinated and non-citrullinated library, to identify potential neo-epitopes and to understand the impact of citrullination on MHC affinity. In parallel we will analyse peripheral blood lymphocytes from 50 T1D patients for immune reactivity against the full library. The large library screen will be conducted...

  3. Development of an epitope panel for consistent identification of antigen-specific T-cells in humans

    DEFF Research Database (Denmark)

    Fløe, Andreas; Løppke, Caroline; Hilberg, Ole

    2017-01-01

    (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection...... a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess......We aimed to establish a panel of MHC–peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from...

  4. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

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

    Directory of Open Access Journals (Sweden)

    Yaakov Maman

    2011-10-01

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

  6. Human CD8+ T cells from TB pleurisy respond to four immunodominant epitopes in Mtb CFP10 restricted by HLA-B alleles.

    Directory of Open Access Journals (Sweden)

    Li Li

    Full Text Available CD8(+ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb infection and identification of CD8(+ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10 from Mtb is a potent CD8(+ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13-15 amino acid (aa peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8(+ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8(+ T cells. The epitope-specific CD8(+ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8(+ T cells shared biased usage of T cell receptor (TCR variable region of β chain (Vβ 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8(+ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8(+ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.

  7. Large-scale analysis of B-cell epitopes on influenza virus hemagglutinin - implications for cross-reactivity of neutralizing antibodies

    DEFF Research Database (Denmark)

    Sun, Jing; Kudahl, Ulrich J.; Simon, Christian

    2014-01-01

    of tens of thousands of HA sequences. The detailed description of B-cell epitopes, measurement of epitope area similarity among different strains, and estimation of antibody neutralizing coverage provide insights into cross-reactivity status of existing nAbs against influenza virus. We have developed...... that share 100% identity with experimentally verified neutralized strains. By cataloging influenza strains and their B-cell epitopes for known bnAbs, our method provides guidance for selection of representative strains for further experimental design. The knowledge of sequences, their B-cell epitopes......Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylactic...

  8. Conservation analysis of dengue virus T-cell epitope-based vaccine candidates using peptide block entropy

    Directory of Open Access Journals (Sweden)

    Lars Ronn Olsen

    2011-12-01

    Full Text Available Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches to assembling broadly covering sets of peptides are commonly based on assembling highly conserved epitopes. Peptide block entropy analysis is a novel approach to assembling sets of broadly covering antigens. Since T-cell epitopes are recognized as peptides rather than individual residues, this method is based on calculating the information content of blocks of peptides from a multiple sequence alignment of homologous proteins rather than individual residues. The block entropy analysis provides broad coverage by variant inclusion, since high frequency may not be the sole determinant of the immunogenic potential of a predicted MHC class I binder. We applied block entropy analysis method to the proteomes of the four serotypes of dengue virus and found 1,551 blocks of 9-mer peptides, which covered all available sequences with five or fewer unique peptides. In contrast, the benchmark study by Khan et al. (2008, resulted in 165 9-mers being determined as conserved. Many of the blocks are located consecutively in the proteins, so connecting these blocks resulted in 78 conserved regions which can be covered with 457 subunit peptides. Of the 1551 blocks of 9-mer peptides, 110 blocks consisted of peptides all predicted to bind to MHC with similar affinity and the same HLA restriction. In total, we identified a pool of 333 peptides as T-cell epitope candidates. This set could form the basis for a broadly neutralizing dengue virus vaccine. The peptide block entropy analysis approach significantly increases the number of conserved peptide regions in comparison to traditional conservation analysis of individual residues. We determined 457 subunit peptides with the capacity to encompass the diversity of all sequenced DENV strains.

  9. Pep-3D-Search: a method for B-cell epitope prediction based on mimotope analysis.

    Science.gov (United States)

    Huang, Yan Xin; Bao, Yong Li; Guo, Shu Yan; Wang, Yan; Zhou, Chun Guang; Li, Yu Xin

    2008-12-16

    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. 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 coefficient (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 similar programs

  10. Immunotherapy with B cell epitopes ameliorates inflammatory responses in Balb/c mice.

    Science.gov (United States)

    Sharma, P; Gaur, S N; Arora, N

    2015-01-01

    Osmotin, a protein from the pathogenesis-related family (PR-5), has been identified as an allergen based on in-silico and in-vitro studies. In the present study, three B cell epitopes of osmotin with single and double amino acid modifications were studied for immunotherapy in a murine model. The single-modification peptides (P-1-1, P-2-1 and P-3-1) and double-modification peptides (P-1-2, P-2-2 and P-3-2) showed significantly lower immunoglobulin (Ig)E binding with patients' sera compared to osmotin (P mice. The sera of mice group treated with peptides showed a significant increase in IgG2a level and a significant decrease in IgE and IgG1 levels (P mice that received peptide immunotherapy showed a shift from a T helper type 2 (Th2) to Th1 type where interferon (IFN)-γ and interleukin (IL)-10 levels were elevated, with a significant increase in groups treated with peptides P-3-1 and P-3-2 (P bronchoalveolar lavage fluid (BALF) in the peptide-treated mice groups. Total cell count and eosinophil count in BALF of the peptide-treated groups was also reduced compared to the phosphate-buffered saline (PBS)-treated group. Lung histology showed a significant reduction in cellular infiltrate in mice treated with P-2-2 and P-3-2 compared to PBS. In conclusion, peptides P-2-2 and P-3-2 lowered inflammatory responses and induced a Th1 response in mice. © 2014 British Society for Immunology.

  11. B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

    Science.gov (United States)

    Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

    2015-10-29

    Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

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

    Science.gov (United States)

    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

  13. Fusion of foreign T-cell epitopes and addition of TLR agonists enhance immunity against Neospora caninum profilin in cattle.

    Science.gov (United States)

    Mansilla, F C; Quintana, M E; Cardoso, N P; Capozzo, A V

    2016-11-01

    We demonstrated recently that immunization with recombinant Neospora caninum profilin (rNcPRO) induces limited protection and a regulatory T-cell response in mice. The aim of this study was to evaluate the immune response elicited by rNcPRO in cattle and assess a strategy to enhance its immunogenicity, combining the addition of T-cell epitopes and immune modulators. We developed a chimeric recombinant profilin fused to functional T-cell epitopes present in the N-terminal sequence of vesicular stomatitis virus (VSV) glycoprotein G (rNcPRO/G). Groups of three cattle were immunized with two doses (2 weeks apart) of rNcPRO or rNcPRO/G formulated with alum hydroxide or a nanoparticulated soya-based adjuvant enriched with Toll-like receptor (TLR) 2 and TLR9 agonists, aimed to tackle the MyD88 pathway (AVECplus). rNcPRO induced only a primary immune response (IgM mediated), while antibodies in rNcPRO/G-vaccinated animals switched to IgG1 after the booster. The vaccine formulated with rNcPRO/G and AVECplus improved the production of systemic IFN-γ and induced long-term recall B-cell responses. Overall, our study provides data supporting the use of T-cell epitopes from VSV glycoprotein G and TLR agonists to enhance and modulate immunity to peptide antigens in bovines, particularly when using small proteins from parasites for which immune responses are usually feeble. © 2016 John Wiley & Sons Ltd.

  14. In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

    Science.gov (United States)

    Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

    2017-07-14

    Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. 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.

    Directory of Open Access Journals (Sweden)

    Jiandong Shi

    Full Text Available 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.

  16. Mapping of epitopes for autoantibodies to the Type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modelling: Overlap of antibody and T-cell determinants

    DEFF Research Database (Denmark)

    A. Dromey, James; Weenink, Sarah M.; Peters, Günther H.J.

    2004-01-01

    IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787–817 and 841–869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide......, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn858, Glu836, and Trp799 reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1...... phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment...

  17. Prediction of T-cell Epitopes for Therapeutic and Prophylactic Vaccines

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby

    2007-01-01

    : The bacteria Mycobacterium tuberculosis, Influenza A virus, HIV, Yellow fever virus, and West Nile virus. For each of the above-mentioned viruses, a number of predicted CTL epitopes was subsequently selected in such a way that they together constitute a broad coverage of the available viral strains. Part IV...

  18. Generation and characterization of recombinant human antibodies specific for native laminin epitopes. Potential application in cancer therapy. Cancer Immunol. Immunother

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Russell, Stephen J.

    2001-01-01

    Laminins are specific cellular regulators that directly and indirectly control activities such as cell attachment and migration, differentiation and polarity, proliferation and apoptosis, and protease expression. Considering the centrality of these issues to tumor progression, the generation of h...

  19. Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A₂ homologue from Bothrops asper snake venom.

    Science.gov (United States)

    Lomonte, Bruno

    2012-10-01

    Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    The affinity with which major histocompatibility complex (MHC) class I molecules bind peptides is instrumental to presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). We analyzed three swine leukocyte antigen (SLA) molecules for complete nonamer peptide-based binding matrices in orde...

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

    Science.gov (United States)

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

    2010-01-01

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

  3. Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Jessica C Graham

    Full Text Available Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX is the approach used to select high affinity aptamers for specific macromolecular targets from among the >10(13 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines.Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX. Using this approach, we identified high affinity aptamers (nanomolar range K(d to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa. We also performed preliminary investigation of the aptamer epitopes and their binding characteristics.Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of

  4. Identification of B-Cell Epitope of Dengue Virus Type 1 and Its Application in Diagnosis of Patients

    OpenAIRE

    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 th...

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

    Science.gov (United States)

    Lu, J; Celis, E

    2000-09-15

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

  6. CD4+ T cell help has an epitope-dependent impact on CD8+ T cell memory inflation during murine cytomegalovirus infection.

    Science.gov (United States)

    Snyder, Christopher M; Loewendorf, Andrea; Bonnett, Elizabeth L; Croft, Michael; Benedict, Chris A; Hill, Ann B

    2009-09-15

    Murine CMV (MCMV) establishes a systemic, low-level persistent infection resulting in the accumulation of CD8(+) T cells specific for a subset of viral epitopes, a process called memory inflation. Although replicating virus is rarely detected in chronically infected C57BL/6 mice, these inflationary cells display a phenotype suggestive of repeated Ag stimulation, and they remain functional. CD4(+) T cells have been implicated in maintaining the function and/or number of CD8(+) T cells in other chronic infections. Moreover, CD4(+) T cells are essential for complete control of MCMV. Thus, we wondered whether CD4(+) T cell deficiency would result in impaired MCMV-specific CD8(+) T cell responses. Here we show that CD4(+) T cell deficiency had an epitope-specific impact on CD8(+) T cell memory inflation. Of the three codominant T cell responses during chronic infection, only accumulation of the late-appearing IE3-specific CD8(+) T cells was substantially impaired in CD4(+) T cell-deficient mice. Moreover, the increased viral activity did not drive increased CD8(+) T cell division or substantial dysfunction in any MCMV-specific population that we studied. These data show that CD4(+) T cell help is needed for inflation of a response that develops only during chronic infection but is otherwise dispensable for the steady state maintenance and function of MCMV-specific CD8(+) T cells.

  7. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    Science.gov (United States)

    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

  8. Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

    Directory of Open Access Journals (Sweden)

    Maria Domina

    Full Text Available We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb epitopes. For this purpose, we used a novel mAb (designated 31E10/E7 directed against Neisserial Heparin-Binding Antigen (NHBA, a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128 in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

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

    DEFF Research Database (Denmark)

    Karlsson, Ingrid; Kløverpris, Henrik; Jensen, Kristoffer Jarlov

    2012-01-01

    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......-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......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...

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

    Directory of Open Access Journals (Sweden)

    Huixin Liu

    2016-01-01

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

  11. P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

    Science.gov (United States)

    Cianfriglia, M; Willingham, M C; Tombesi, M; Scagliotti, G V; Frasca, G; Chersi, A

    1994-01-02

    A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

  12. Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects.

    Science.gov (United States)

    Vijayamahantesh; Amit, Ajay; Dikhit, Manas R; Singh, Ashish K; Venkateshwaran, T; Das, V N R; Das, Pradeep; Bimal, Sanjiva

    2017-06-01

    Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8 + T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8 + T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8 + T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3'-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02 + visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8 + cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02 + VL subjects. Thus, the CD8 + T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis. Copyright © 2017. Published by Elsevier Masson SAS.

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

    NARCIS (Netherlands)

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

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

  14. T cell immunity to Zika virus targets immunodominant epitopes that show cross-reactivity with other Flaviviruses.

    Science.gov (United States)

    Reynolds, C J; Suleyman, O M; Ortega-Prieto, A M; Skelton, J K; Bonnesoeur, P; Blohm, A; Carregaro, V; Silva, J S; James, E A; Maillère, B; Dorner, M; Boyton, R J; Altmann, D M

    2018-01-12

    Zika virus (ZIKV) Infection has several outcomes from asymptomatic exposure to rash, conjunctivitis, Guillain-Barré syndrome or congenital Zika syndrome. Analysis of ZIKV immunity is confounded by the fact that several related Flaviviruses infect humans, including Dengue virus 1-4, West Nile virus and Yellow Fever virus. HLA class II restricted T cell cross-reactivity between ZIKV and other Flaviviruses infection(s) or vaccination may contribute to protection or to enhanced immunopathology. We mapped immunodominant, HLA class II restricted, CD4 epitopes from ZIKV Envelope (Env), and Non-structural (NS) NS1, NS3 and NS5 antigens in HLA class II transgenic mice. In several cases, ZIKV primed CD4 cells responded to homologous sequences from other viruses, including DENV1-4, WNV or YFV. However, cross-reactive responses could confer immune deviation - the response to the Env DENV4 p1 epitope in HLA-DR1 resulted in IL-17A immunity, often associated with exacerbated immunopathogenesis. This conservation of recognition across Flaviviruses, may encompass protective and/or pathogenic components and poses challenges to characterization of ZIKV protective immunity.

  15. Identification of novel rabbit hemorrhagic disease virus B-cell epitopes and their interaction with host histo-blood group antigens.

    Science.gov (United States)

    Song, Yanhua; Wang, Fang; Fan, Zhiyu; Hu, Bo; Liu, Xing; Wei, Houjun; Xue, Jiabin; Xu, Weizhong; Qiu, Rulong

    2016-02-01

    Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.

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

    Directory of Open Access Journals (Sweden)

    Felici Franco

    2007-12-01

    Full Text Available Abstract 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.

  17. Relationship between Poor Immunogenicity of HLA-A2-Restricted Peptide Epitopes and Paucity of Naïve CD8+ T-Cell Precursors in HLA-A2-Transgenic Mice

    OpenAIRE

    Choi, Yoon Seok; Lee, Dong Ho; Shin, Eui-Cheol

    2014-01-01

    We examined the immunogenicity of H-2 class I-restricted and HLA-A2-restricted epitopes through peptide immunization of HLA-A2-transgenic mice that also express mouse H-2 class I molecules. All four of the tested epitopes restricted by H-2 class I robustly elicited T-cell responses, but four of seven epitopes restricted by HLA-A2 did not induce T-cell responses, showing that HLA-A2-restricted peptide epitopes tend to be poorly immunogenic in HLA-A2-transgenic mice. This finding was confirmed ...

  18. Identification of the impact on T- and B- cell epitopes of human papillomavirus type-16 E6 and E7 variant in Southwest China.

    Science.gov (United States)

    Chenzhang, Yuwei; Wen, Qiang; Ding, Xianping; Cao, Man; Chen, Zuyi; Mu, Xuemei; Wang, Tao

    2017-01-01

    Cervical cancers almost are infected by human papillmavirus (HPV), encoding E6 and E7 oncoproteins which are regard as ideal targets on the mechanism of this disease and development of vaccines. HLA (human leukocyte antigen) participates in the local immune response to prevent tumor invasion and progression. But due to highly polymorphism of HLA, prediction shows its importance in this study. More effective immunoinformatics was used for predicting epitopes from HPV-16 E6 and E7, including T- and B-cell epitopes. Eight substitutions are detected. Specifically speaking, for HLA-I, HLA-A*33:03 (26), HLA-B*13:01 (14), HLA-C*03:02 (5) for E6 and HLA-A*02:01 (6), HLA-B*40:01 (5), HLA-C*03:04 (4) for E7 are most frequency. Epitope 41-48 EVYDFAFR for HLA-A*33:03 (0.1) for E6 has best binding affinity, as well as HLA*02:01 and HLA-B*40:01 (0.2) for E7. The mutations of D25E and L83V of E6 and N29S of E7 produce new epitopes, and the percentile values change with them. For HLA-II, seventeen epitopes in the reference at percentile value from 0.22 to 4.76, while in variant from 0.22 to 4.96. For the B-cell epitopes, three most potent epitopes for E6 were listed, and N29S lead the growth of score from 0.81 to 0.83. In summary, E6 40-55 REVYDFAFRDLCIVYR and E7 11-22 YMLDLQPETTDL are the important regions, containing the majority of predicted epitopes. E6 72-83 for HLA-A*02:01 and E6 74-84 for HLA-B*15:02 maybe are the new direct for therapeutic vaccine aimed at L83V variants. HLA-DRB1*15:02 is better binder with T cell in our HLA class II. It is a systematic, detail recognition for T- and B-cell epitopes of HPV-16 E6 and E7 from Southwest China, which may be helpful to design vaccines specifically for women in Southwest China and testing methods specifically for this region. The results of our study may contribute to future researches on vaccines improvement, or screening methods for a particular population. Copyright © 2016 European Federation of Immunological Societies

  19. Diverse patterns of T-cell response against multiple newly identified human Y chromosome-encoded minor histocompatibility epitopes.

    Science.gov (United States)

    Ofran, Yishai; Kim, Haesook T; Brusic, Vladimir; Blake, Loren; Mandrell, Michael; Wu, Catherine J; Sarantopoulos, Stefanie; Bellucci, Roberto; Keskin, Derin B; Soiffer, Robert J; Antin, Joseph H; Ritz, Jerome

    2010-03-01

    Donor T cells respond to minor histocompatibility antigens (mHA), resulting in both graft-versus-host disease and graft versus leukemia after allogeneic hematopoietic stem cell transplantation. Because relatively few mHAs are known, we developed a new approach to predict and subsequently validate candidate mHA. We developed an algorithm based on genetic disparities between Y chromosome-encoded and X chromosome-encoded proteins and known requirements for binding to HLA class I molecules to predict Y chromosome-derived, HLA A*0201-restricted peptides (HY) and ranked peptides based on potential immunogenicity. We evaluated T-cell responses to 41 candidate peptides in 28 male recipients with female donors (FM), 22 male recipients with male donors (MM), and 26 normal individuals. All patients and donors were HLA A*0201 positive. Thirteen peptides derived from five proteins elicited significantly greater T-cell responses in FM patients compared with MM patients and in normal females compared with normal males. Six peptides were more immunogenic than the only previously known HLA A*0201-restricted Y-encoded mHA. Twenty-seven of 28 FM patients responded to at least one HY peptide, but despite a common Y chromosome mismatch and expression of HLA A*0201, each patient responded to a unique set of peptides. Novel HLA A*0201-restricted HY epitopes can be predicted and validated in patients after allogeneic hematopoietic stem cell transplantation. Highly diverse patterns of T-cell response against these epitopes have been identified. Prospective monitoring of responses to large panels of immunogenic peptides can facilitate the identification of clinically relevant targets of graft-versus-host disease and graft versus leukemia.

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

    OpenAIRE

    Stranzl, Thomas; Larsen, Mette V.; Lund, Ole; Nielsen, Morten; Brunak, Søren

    2012-01-01

    Several studies have shown that cancers actively regulate alternative splicing. Altered splicing mechanisms in cancer lead to cancer-specific transcripts different from the pool of transcripts occurring only in healthy tissue. At the same time, altered presentation of HLA class I epitopes is frequently observed in various types of cancer. Down-regulation of genes related to HLA class I antigen processing has been observed in several cancer types, leading to fewer HLA class I antigens on the c...

  1. Molecular characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and impact of T-cell epitope mutations on HLA recognition (ANRS 12159.

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    Estibaliz Lazaro

    Full Text Available BACKGROUND: To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding. METHODS: We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores and HLA binding (MHC score. RESULTS: All sequences clustered with CRF01_AE. HLA class I genotyping showed the predominance of Asian alleles as A*11:01 and B*46:01 with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9 versus 2 (±0.7 in non-divergent ones (p<0.0001. CONCLUSIONS: Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area.

  2. Presentation of a Conserved Adenoviral Epitope on HLA-C*0702 Allows Evasion of Natural Killer but Not T Cell Responses.

    Science.gov (United States)

    Keib, Anna; Günther, Patrick S; Faist, Benjamin; Halenius, Anne; Busch, Dirk H; Neuenhahn, Michael; Jahn, Gerhard; Dennehy, Kevin M

    2017-04-01

    Infection with adenovirus is a major cause of infectious mortality in children following hematopoietic stem-cell transplantation. While adoptive transfer of epitope-specific T cells is a particularly effective therapeutic approach, there are few suitable adenoviral peptide epitopes described to date. Here, we describe the adenoviral peptide epitope FRKDVNMVL from hexon protein, and its variant FRKDVNMIL, that is restricted by human leukocyte antigen (HLA)-C*0702. Since HLA-C*0702 can be recognized by both T cells and natural killer (NK) cells, we characterized responses by both cell types. T cells specific for FRKDVNMVL were detected in peripheral blood mononuclear cells expanded from eight of ten healthy HLA-typed donors by peptide-HLA multimer staining, and could also be detected by cultured interferon γ ELISpot assays. Surprisingly, HLA-C*0702 was not downregulated during infection, in contrast to the marked downregulation of HLA-A*0201, suggesting that adenovirus cannot evade T cell responses to HLA-C*0702-restricted peptide epitopes. By contrast, NK responses were inhibited following adenoviral peptide presentation. Notably, presentation of the FRKDVNMVL peptide enhanced binding of HLA-C*0702 to the inhibitory receptor KIR2DL3 and decreased NK cytotoxic responses, suggesting that adenoviruses may use this peptide to evade NK responses. Given the immunodominance of FRKDVNMVL-specific T cell responses, apparent lack of HLA-C*0702 downregulation during infection, and the high frequency of this allotype, this peptide epitope may be particularly useful for adoptive T cell transfer therapy of adenovirus infection.

  3. A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen.

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    Rona Y Zhao

    Full Text Available 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-1(88-96 (LEFYLAMPF and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165 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-1(88-96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165. On the other hand, NY-ESO-1(157-165 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 A(26-35; whereas NY-ESO-1(88-96 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-1(88-96 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-1(88-96 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.

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

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    Salentijn Elma MJ

    2012-06-01

    Full Text Available Abstract 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

  5. Phage displayed short peptides against cells of Candida albicans demonstrate presence of species, morphology and region specific carbohydrate epitopes.

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    Soshee Anandakumar

    Full Text Available Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

  6. Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model.

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    Simon H Apte

    Full Text Available Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+ and/or CD8(+ T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+ T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+ T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

  7. Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model.

    Science.gov (United States)

    Apte, Simon H; Groves, Penny L; Skwarczynski, Mariusz; Fujita, Yoshio; Chang, Chenghung; Toth, Istvan; Doolan, Denise L

    2012-01-01

    Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+) and/or CD8(+) T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+) T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+) T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

  8. Immune hierarchy among HIV-1 CD8+ T cell epitopes delivered by dendritic cells depends on MHC-I binding irrespective of mode of loading and immunization in HLA-A*0201 mice

    DEFF Research Database (Denmark)

    Kloverpris, Henrik N; Karlsson, Ingrid; Thorn, Mette

    2009-01-01

    Recent human immunodeficiency virus type 1 (HIV-1) vaccination strategies aim at targeting a broad range of cytotoxic T lymphocyte (CTL) epitopes from different HIV-1 proteins by immunization with multiple CTL epitopes simultaneously. However, this may establish an immune hierarchical response......, where the immune system responds to only a small number of the epitopes administered. To evaluate the feasibility of such vaccine strategies, we used the human leukocyte antigen (HLA)-A*0201 transgenic (tg) HHD murine in vivo model and immunized with dendritic cells pulsed with seven HIV-1-derived HLA......-gamma)-producing CD8(+) T cells, mainly focused on two of seven administered epitopes. The magnitude of individual T-cell responses induced by immunization with multiple peptides correlated with their individual immunogenicity that depended on major histocompatibility class I binding and was not influenced by mode...

  9. Arabinogalactan-protein epitope Gal4 is differentially regulated and localized in cell lines of hybrid fir (Abies alba x Abies cephalonica) with different embryogenic and regeneration potential.

    Science.gov (United States)

    Samaj, Jozef; Salaj, Terézia; Matúsová, Radoslava; Salaj, Ján; Takác, Tomás; Samajová, Ol'ga; Volkmann, Dieter

    2008-02-01

    Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network (ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba x Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing beta-(1-->6)-galactotetraosyl group as a first molecular component of ECMSN covering embryogenic cells in gymnosperms.

  10. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

    Science.gov (United States)

    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.

  11. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

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    Ryan D Pardy

    2017-02-01

    Full Text Available 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.

  12. CD4+ T cell autoimmunity to hypocretin/orexin and cross-reactivity to a 2009 H1N1 influenza A epitope in narcolepsy

    DEFF Research Database (Denmark)

    De la Herrán-Arita, Alberto K; Kornum, Birgitte Rahbek; Mahlios, Josh

    2013-01-01

    the wake-promoting neuropeptide hypocretin (HCRT) (orexin). We identified two DQ0602-binding HCRT epitopes, HCRT56-68 and HCRT87-99, that activated a subpopulation of CD4(+) T cells in narcolepsy patients but not in DQ0602-positive healthy control subjects. Because of the established association...

  13. Identification of Rotavirus VP6-Specific CD4+ T Cell Epitopes in a G1P[8] Human Rotavirus-Infected Rhesus Macaque

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    Wei Zhao

    2008-01-01

    Full Text Available A non-human primate model was used to evaluate its potential for identification of rotavirus viral protein 6 (VP6 CD4+ T cell epitopes. Four juvenile rhesus macaques were inoculated with a mixed inoculum (G1P[8] and G9P[8] of human rotaviruses. Infection accompanied by G1P[8] shedding was achieved in the two macaques that had no rotavirus immunoglobulin A (IgA in plasma. To measure the interferon gamma (IFN-γ and tumor necrosis factor (TNF anti-viral cytokines produced by peripheral CD4+ cells that recognize VP6 epitopes, whole blood cells from one infected macaque were stimulated in vitro with VP6 peptides. Stimulation with peptide pools derived from the simian rotavirus VP6 161–395 region revealed reactivity of CD4+ T cells with the VP6 281–331 domain. A VP6 301–315 region was identified as the epitope responsible for IFN-γ production while a broader VP6 293–327 domain was linked to TNF production. These results suggest that human rotavirus-infected macaques can be used for identification of additional epitopes and domains to address specific questions related to the development of pediatric vaccines.

  14. Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets.

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    S K Rosendahl Huber

    Full Text Available Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP, polymerase basic protein 1 (PB1 and matrix protein 1 (M1. C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.

  15. Analysis of Nanobody-Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.

    Science.gov (United States)

    Früholz, Simone; Pimpl, Peter

    2017-01-01

    Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins.Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.

  16. Structural basis for clonal diversity of the human T-cell response to a dominant influenza virus epitope

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xinbo; Chen, Guobing; Weng, Nan-ping; Mariuzza, Roy A. (NIH); (Maryland-BI)

    2017-09-20

    Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8+ cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/β chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ~40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50) expressing the TRAV13-1/TRBV27 gene combination bound to GIL–HLA-A2 to 1.7 Å resolution. Comparison of the F50–GIL–HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL–HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide–MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50–GIL–HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide–MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.

  17. Identification of CD4+ T-cell Epitopes on Mycobacterium Tuberculosis- Secreted MPB51 Protein in C57BL/6 Mice

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    A.R. Rafiei

    2006-01-01

    Full Text Available Introduction & Objective: Both CD4+ type 1 helper (Th1 cells and CD8+ T cells play effective roles in protection against Mycobacterium tuberculosis infection. DNA vaccine encoding MPB51 can induce Th1-type immune responses and protective immunity upon challenge with M.tuberculosis. This study address to identify T-cell immunodominant epitopes on MPB51 in C57BL/6 mice.Materials & Methods : We cloned DNA encoding MPB51 molecule in pCI plasmid. After constructing MPB51 DNA-covered gold cartridge, C57BL/6 mice were immunized by using a gene gun system. Two weeks after the last immunization, the immune spleen cells were cultured in the presence of a synthetic overlapping library peptides covering the mature MPB51 sequence or medium alone. Intracellular and cell culture supernatant gamma interferon (IFN- production was analyzed using flow cytometry and ELISA, respectively.Results : Mapping of T-cell epitopes on MPB51 molecule was performed in the spleen lymphocytes restimulated by 20-mer overlapping synthetic peptides of mature MPB51 sequence. Flow cytometric analysis with intracellular IFN- and the T-cell phenotype revealed that P171-190 and P191-210 peptides contain immunodominant CD4+ T-cell epitopes. Further analysis by using T-cell subset depletion and serial peptide dilution revealed that P171 and p191 are H2-Ab-restricted dominant and subdominant CD4+ T cell epitopes, respectively. Conclusion: This study proved that vaccination with plasmid DNA encoding M. tuberculosis-secreted MPB51 protein not only induce CD4+ T cells immune response but also is an appropriate method for identifying immunogenic peptides.

  18. Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

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    Peter C. Soema

    2018-03-01

    Full Text Available Influenza peptide antigens coding for conserved T cell epitopes have the capacity to induce cross-protective influenza-specific immunity. Short peptide antigens used as a vaccine, however, often show poor immunogenicity. In this study, we demonstrate that whole-inactivated influenza virus (WIV acts as an adjuvant for influenza peptide antigens, as shown by the induction of peptide-specific CD8+ T cells in HLA-A2.1 transgenic mice upon vaccination with the influenza-M1-derived GILGFVFTL peptide (GIL, formulated with WIV. By screening various concentrations of GIL and WIV, we found that both components contributed to the GIL-specific T cell response. Whereas co-localization of the peptide antigen and WIV adjuvant was found to be important, neither physical association between peptide and WIV nor fusogenic activity of WIV were relevant for the adjuvant effect of WIV. We furthermore show that WIV may adjuvate T cell responses to a variety of peptides, using pools of either conserved wild-type influenza peptides or chemically altered peptide ligands. This study shows the potential of WIV as an adjuvant for influenza peptides. The simple formulation process and the solid safety record of WIV make this an attractive adjuvant for T cell peptides, and may also be used for non-influenza antigens.

  19. A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets

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    Kenneth R. Boheler

    2014-07-01

    Full Text Available Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs and induced PSCs (hiPSCs. Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

  20. Fine T cell receptor repertoire analysis of spinal cord T cells responding to the major and minor epitopes of myelin basic protein during rat autoimmune encephalomyelitis.

    Science.gov (United States)

    Matsumoto, Y; Jee, Y; Sugisaki, M; Kim, G; Tanuma, N

    2000-01-01

    Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectra-typing. It was revealed that the oligoclonal expansion of Vbeta2, Vbeta8.2, and Vbeta17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68-88 sequence, whereas immunization with a peptide containing a minor epitope induced Vbeta10 expansion. Immunization with rat MBP induced much broader TCR Vbeta expansion (all of the above Vbetas plus Vbeta3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy.

  1. CD8 T cells control cytomegalovirus latency by epitope-specific sensing of transcriptional reactivation.

    Science.gov (United States)

    Simon, Christian O; Holtappels, Rafaela; Tervo, Hanna-Mari; Böhm, Verena; Däubner, Torsten; Oehrlein-Karpi, Silke A; Kühnapfel, Birgit; Renzaho, Angélique; Strand, Dennis; Podlech, Jürgen; Reddehase, Matthias J; Grzimek, Natascha K A

    2006-11-01

    During murine cytomegalovirus (mCMV) latency in the lungs, most of the viral genomes are transcriptionally silent at the major immediate-early locus, but rare and stochastic episodes of desilencing lead to the expression of IE1 transcripts. This low-frequency but perpetual expression is accompanied by an activation of lung-resident effector-memory CD8 T cells specific for the antigenic peptide 168-YPHFMPTNL-176, which is derived from the IE1 protein. These molecular and immunological findings were combined in the "silencing/desilencing and immune sensing hypothesis" of cytomegalovirus latency and reactivation. This hypothesis proposes that IE1 gene expression proceeds to cell surface presentation of the IE1 peptide by the major histocompatibility complex (MHC) class I molecule L(d) and that its recognition by CD8 T cells terminates virus reactivation. Here we provide experimental evidence in support of this hypothesis. We generated mutant virus mCMV-IE1-L176A, in which the antigenic IE1 peptide is functionally deleted by a point mutation of the C-terminal MHC class I anchor residue Leu into Ala. Two revertant viruses, mCMV-IE1-A176L and the wobble nucleotide-marked mCMV-IE1-A176L*, in which Leu is restored by back-mutation of Ala codon GCA into Leu codons CTA and CTT, respectively, were constructed. Pulmonary latency of the mutant virus was found to be associated with an increased prevalence of IE1 transcription and with events of IE3 transactivator splicing. In conclusion, IE1-specific CD8 T cells recognize and terminate virus reactivation in vivo at the first opportunity in the reactivated gene expression program. The perpetual gene expression and antigen presentation might represent the driving molecular force in CMV-associated immunosenescence.

  2. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    Science.gov (United States)

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

  3. Prediction of antigenic epitopes and MHC binders of neurotoxin ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... learning techniques such as Support Vector Machine (SVM) and Artificial Neural Network (ANN). This method has been trained and tested on non-redundant dataset of T cell epitopes and non-epitopes that includes 1137 experimen- tally proven MHC class 1 restricted T cell epitopes. (Bhasin and Raghava ...

  4. Myeloid dendritic cells loaded with dendritic tandem multiple antigenic telomerase reverse transcriptase (hTERT) epitope peptides: a potentially promising tumor vaccine.

    Science.gov (United States)

    Niu, Bai-lin; Du, Hui-min; Shen, Hua-ping; Lian, Zheng-rong; Li, Jin-zheng; Lai, Xing; Wei, Si-dong; Zou, Li-quan; Gong, Jian-ping

    2012-05-14

    Human telomerase reverse transcriptase (hTERT) has been identified as an ideal tumor-associated antigen (TAA). Use of a synthetic hTERT epitope peptide to pulse dendritic cells can induce autologous T cell anti-tumor immune responses, but such responses induced by a single epitope peptide have been shown to be weak and a narrow-spectrum. Here, we designed dendritic tandem multiple antigenic peptides (MAPs) containing the following three hTERT epitope peptides: I540, V461 and L766, which are HLA-A*02-, HLA-A*24- and HLA-RDB1*04/11/15-restricted, respectively. The MAPs and their three single-epitope peptides were obtained through solid-phase synthesis. Healthy volunteers that were HLA-A*02(+)/HLA-DRB1*04(+) and HLA-A*24(+)/HLA-DRB1*15(+) were recruited. Myeloid dendritic cells were isolated by magnetic activated cell sorting and were divided into a MAP-stimulated group (MAP-DC), a group in which the three epitope peptides were mixed and used to stimulate the DCs (MixP-DC) and a no peptide-stimulated group (NoP-DC, control group). All of the DCs were cultured in serum-free medium, pulsed with the corresponding peptides on the 3rd, 5th and 7th days, and co-cultured with autologous lymphocytes when they were mature. The related cytokines were measured via ELISA. The killing effects of cytotoxic T lymphocytes (CTLs) on SW480/A549 tumor cells expressing HLA-A*02(+), HepG2/SMMC-7721 cells expressing HLA-A*24(+) and SKOV3 cells negative for HLA-A*02/A*24 were detected by flow cytometry. Our results indicated that the CTLs induced by the MAP-DCs had the greatest anti-tumor effect. Therefore, the dendritic tandem multiple antigenic hTERT epitope peptides combined with MDCs may represent a powerful, broad-spectrum anti-tumor vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. The Immune Epitope Database 2.0

    DEFF Research Database (Denmark)

    Hoof, Ilka; Vita, R; Zarebski, L

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive...... immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course...

  6. Intraoral administration of a T-cell epitope peptide induces immunological tolerance in Cry j 2-sensitized mice.

    Science.gov (United States)

    Yoshitomi, Tomomi; Nakagami, Yasuhiro; Hirahara, Kazuki; Taniguchi, Yoshifumi; Sakaguchi, Masahiro; Yamashita, Makoto

    2007-08-01

    Sublingual immunotherapy using allergen-derived peptides is feasible as a novel specific immunotherapy, but its efficacy has not yet been demonstrated in either humans or animals. In addition, it remains obscure whether the oral immune system is involved in the mechanism of sublingual immunotherapy. Here, we show that the intraoral administration of the T-cell epitope peptide P2-246-259 derived from Cry j 2, a major Japanese cedar (Cryptomeria japonica) pollen allergen, to Cry j 2-sensitized mice induces immunological tolerance, and that ex vivo lymph node cell proliferation to P2-246-259 and Cry j 2 was inhibited. In addition, intraoral administration was shown to be superior to intragastric administration in terms of tolerance induction, suggesting that the oral immune system contributes to the induction of immunological tolerance. Therefore, the significant efficacy of sublingual immunotherapy using a peptide on allergen-specific T-cells was demonstrated in animals, and this may be potentiated by the oral mucosal immune system. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

  7. Fast generation of dendritic cells

    DEFF Research Database (Denmark)

    Kvistborg, P; Bøgh, Marie; Pedersen, A W

    2009-01-01

    Dendritic cells (DC) are potent antigen presenting cells capable of inducing immune responses. DC are widely used as vaccine adjuvant in experimental clinical settings. DC-based vaccines are normally generated using a standard 8day DC protocol (SDDC). In attempts to shorten the vaccine production...... SDDC to the IL-10 inducing stimulus of TLR ligands (R848 and LPS). Thus to determine the clinical relevance of fast DC protocols in cancer settings, small phase I trials should be conducted monitoring regulatory T cells carefully....

  8. Identification of three novel B-cell epitopes of VMH protein from Vibrio mimicus by screening a phage display peptide library.

    Science.gov (United States)

    Xiao, Ning; Cao, Ji; Zhou, Hao; Ding, Shu-Quan; Kong, Ling-Yan; Li, Jin-Nian

    2016-12-01

    Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children.

    Science.gov (United States)

    Chawansuntati, Kriangkrai; Aurpibul, Linda; Wipasa, Jiraprapa

    2015-09-11

    The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A vaccine encoding conserved promiscuous HIV CD4 epitopes induces broad T cell responses in mice transgenic to multiple common HLA class II molecules.

    Directory of Open Access Journals (Sweden)

    Susan Pereira Ribeiro

    Full Text Available Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, "promiscuous" (multiple HLA-DR-binding B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8. Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

  11. Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

    DEFF Research Database (Denmark)

    Claesson, M H; Endel, B; Ulrik, J

    1994-01-01

    binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2b, H-2d, as well as H-2k cells by FACS analysis. The MoAbs stained...... to two beta 2m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous beta 2m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human......-S cells were cultured at 37 degrees C. In total, these observations suggest that the MoAbs recognize conformational, presumably beta 2m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas...

  12. Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies.

    Directory of Open Access Journals (Sweden)

    Rong-Hong Hua

    Full Text Available Japanese encephalitis virus (JEV non-structural protein 1 (NS1 contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA, five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues (5AIDITRK(11, (72RDELNVL(78, (251KSKHNRREGY(260, (269DENGIVLD(276, and (341DETTLVRS(348. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.

  13. Reversal of tolerance induced by transplantation of skin expressing the immunodominant T cell epitope of rat type II collagen entitles development of collagen-induced arthritis but not graft rejection

    DEFF Research Database (Denmark)

    Bäcklund, Johan; Treschow, Alexandra; Firan, Mihail

    2002-01-01

    Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I...... collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a naïve immune system would lead to T cell priming and graft...

  14. Maritime Fuel Cell Generator Project.

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, Joseph William [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2017-07-01

    Fuel costs and emissions in maritime ports are an opportunity for transportation energy efficiency improvement and emissions reduction efforts. Ocean-going vessels, harbor craft, and cargo handling equipment are still major contributors to air pollution in and around ports. Diesel engine costs continually increase as tighter criteria pollutant regulations come into effect and will continue to do so with expected introduction of carbon emission regulations. Diesel fuel costs will also continue to rise as requirements for cleaner fuels are imposed. Both aspects will increase the cost of diesel-based power generation on the vessel and on shore. Although fuel cells have been used in many successful applications, they have not been technically or commercially validated in the port environment. One opportunity to do so was identified in Honolulu Harbor at the Young Brothers Ltd. wharf. At this facility, barges sail regularly to and from neighbor islands and containerized diesel generators provide power for the reefers while on the dock and on the barge during transport, nearly always at part load. Due to inherent efficiency characteristics of fuel cells and diesel generators, switching to a hydrogen fuel cell power generator was found to have potential emissions and cost savings.

  15. Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

    Science.gov (United States)

    Dias-Lopes, C; Felicori, L; Rubrecht, L; Cobo, S; Molina, L; Nguyen, C; Galéa, P; Granier, C; Molina, F; Chávez-Olortegui, C

    2014-04-11

    We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

    Science.gov (United States)

    Sancho, Ana I; Wallner, Michael; Hauser, Michael; Nagl, Birgit; Himly, Martin; Asam, Claudia; Ebner, Christof; Jahn-Schmid, Beatrice; Bohle, Barbara; Ferreira, Fatima

    2017-01-01

    Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy. Domains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT. The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected. Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

  17. T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

    Directory of Open Access Journals (Sweden)

    Ana I Sancho

    Full Text Available Ragweed (Ambrosia artemisiifolia and mugwort (Artemisia vulgaris are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1 and mugwort pollen (Art v 6 for immunotherapy.Domains of Amb a 1 (Amb a 1α and Art v 6 (Art v 6α and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT.The IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected.Accumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

  18. A targeted LC-MS strategy for low-abundant HLA class I-presented peptide detection identifies novel human papillomavirus T-cell epitopes.

    Science.gov (United States)

    Blatnik, Renata; Mohan, Nitya; Bonsack, Maria; Falkenby, Lasse G; Hoppe, Stephanie; Josef, Kathrin; Steinbach, Alina; Becker, Sara; Nadler, Wiebke M; Rucevic, Marijana; Larsen, Martin R; Salek, Mogjiborahman; Riemer, Angelika B

    2018-03-30

    For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes, either of viral origin or mutation-derived, are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC-MS 3 -based targeted detection of low-abundant HLA class-I presented peptides from transformed cells. We used human papillomavirus (HPV) as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. Our approach included preselection of target antigen-derived peptides by in silico predictions and in vitro binding assays. We tailored the peptide purification process to minimize contaminants after immunoprecipitation of HLA-peptide complexes, while keeping high isolation yields of low-abundant target peptides. The subsequent targeted LC-MS 3 detection allowed for increased sensitivity, which resulted in successful detection of the previously described HLA-A2-restricted epitope E7 11-19 and 10 additional E7-derived peptides on the surface of HPV16-transformed cells. T-cell reactivity was shown for all of the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. Thus, it is suitable for validating even low-abundant candidate epitopes to be true immunotherapy targets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. Simultaneous immunisation with a Wilms' tumour 1 epitope and its ubiquitin fusions results in enhanced cell mediated immunity and tumour rejection in C57BL/6 mice.

    Science.gov (United States)

    Eslami, Nasir Saeedi; Shokrgozar, Mohammad Ali; Mousavi, Asadollah; Azadmanesh, Kayhan; Nomani, Alireza; Apostolopoulos, Vasso; Day, Stephanie; Amanzadeh, Amir; Alimohammadian, Mohammad Hossein

    2012-07-01

    Protein fusion to ubiquitin results in its targeting to proteasome and processing through MHC class I pathway. We used this approach to induce cytotoxic T lymphocyte (CTL) response against a MHC class I epitope. Therefore, two known proteasome targeting systems, "ubiquitin fusion degradation" (UFD) and "N-end rule", were used to immunise C57BL/6 mice. Two plasmids encoding an epitope from Wilms' Tumour 1 (WT1-126), fused N-terminally to ubiquitin, were constructed. They were designated as "pUbVVPT" and "pUbGRPT", targeting the fused epitope to UFD and N-end pathways, respectively. A plasmid encoding WT1-126 without ubiquitin fusion (pPT) was also constructed as control. Three mice groups were immunised using these constructs (UGR, UVV and PT groups). Two other groups received mixed immunisations of pUbVVPT or pUbGRPT plus pPT plasmids (UVV+PT and UGR+PT). All mice received a WT1-126 peptide booster. Lymphoproliferative responses following stimulation with WT1-126 were observed in all immunisation groups, with mice receiving the mixture of plasmids eliciting the highest proliferation (UVV+PT>UGR+PT>PT). Moreover, In vivo cytotoxicity assay results revealed highest specific lysis of target cells in UVV+PT group. Tumour growth was decreased in all immunised groups, and was completely abrogated in UGR+PT group. In addition, T(H)1 type cytokines patterns were detected from all immunised groups and WT1-126-specific IFNγ producing lymphocytes were developed in them. These results suggest that the delivery of ubiquitin-fused epitopes along with epitopes alone can be used to optimise the effect of DNA vaccines on the induction of anti-tumour immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8+ T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

    Science.gov (United States)

    Khan, Arif A.; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P.; Pham, Thanh T.; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M.; Nesburn, Anthony B.

    2015-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8+ T cells which responded mainly to gB342–350 and gB561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107a/b, granzyme B, and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17–25 and gB183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8+ T cells (TEM

  1. Pectic-β(1,4)-galactan, extensin and arabinogalactan–protein epitopes differentiate ripening stages in wine and table grape cell walls

    Science.gov (United States)

    Moore, John P.; Fangel, Jonatan U.; Willats, William G. T.; Vivier, Melané A.

    2014-01-01

    Background and Aims Cell wall changes in ripening grapes (Vitis vinifera) have been shown to involve re-modelling of pectin, xyloglucan and cellulose networks. Newer experimental techniques, such as molecular probes specific for cell wall epitopes, have yet to be extensively used in grape studies. Limited general information is available on the cell wall properties that contribute to texture differences between wine and table grapes. This study evaluates whether profiling tools can detect cell wall changes in ripening grapes from commercial vineyards. Methods Standard sugar analysis and infra-red spectroscopy were used to examine the ripening stages (green, véraison and ripe) in grapes collected from Cabernet Sauvignon and Crimson Seedless vineyards. Comprehensive microarray polymer profiling (CoMPP) analysis was performed on cyclohexanediaminetetraacetic acid (CDTA) and NaOH extracts of alcohol-insoluble residue sourced from each stage using sets of cell wall probes (mAbs and CBMs), and the datasets were analysed using multivariate software. Key Results The datasets obtained confirmed previous studies on cell wall changes known to occur during grape ripening. Probes for homogalacturonan (e.g. LM19) were enriched in the CDTA fractions of Crimson Seedless relative to Cabernet Sauvignon grapes. Probes for pectic-β-(1,4)-galactan (mAb LM5), extensin (mAb LM1) and arabinogalactan proteins (AGPs, mAb LM2) were strongly correlated with ripening. From green stage to véraison, a progressive reduction in pectic-β-(1,4)-galactan epitopes, present in both pectin-rich (CDTA) and hemicellulose-rich (NaOH) polymers, was observed. Ripening changes in AGP and extensin epitope abundance also were found during and after véraison. Conclusions Combinations of cell wall probes are able to define distinct ripening phases in grapes. Pectic-β-(1,4)-galactan epitopes decreased in abundance from green stage to véraison berries. From véraison there was an increase in abundance of

  2. Development of a POC test for TB based on multiple immunodominant epitopes of M. tuberculosis specific cell-wall proteins.

    Directory of Open Access Journals (Sweden)

    Jesus M Gonzalez

    Full Text Available The need for an accurate, rapid, simple and affordable point-of-care (POC test for Tuberculosis (TB that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28-85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC

  3. Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues

    DEFF Research Database (Denmark)

    Arentz-Hansen, Helene; McAdam, Stephen N; Molberg, Øyvind

    2002-01-01

    BACKGROUND & AIMS: Celiac disease is a gluten-induced enteropathy that shows a strong association with HLA-DQ2 and -DQ8. Gluten-specific T cells, invariably restricted by DQ2 or DQ8, can be isolated from celiac lesions. Such gut-derived T cells have a preference for recognition of gluten that has...

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, ...

  5. Linear B-cell epitope mapping of MAPK3 and MAPK4 from Leishmania braziliensis: implications for the serodiagnosis of human and canine leishmaniasis.

    Science.gov (United States)

    Menezes-Souza, Daniel; de Oliveira Mendes, Tiago Antônio; de Araújo Leão, Ana Carolina; de Souza Gomes, Matheus; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella Castanheira

    2015-02-01

    The correct and early identification of humans and dogs infected with Leishmania are key steps in the control of leishmaniasis. Additionally, a method with high sensitivity and specificity at low cost that allows the screening of a large number of samples would be extremely valuable. In this study, we analyzed the potential of mitogen-activated protein kinase 3 (MAPK3) and mitogen-activated protein kinase 4 (MAPK4) proteins from Leishmania braziliensis to serve as antigen candidates for the serodiagnosis of human visceral and tegumentary leishmaniasis, as well as canine visceral disease. Moreover, we mapped linear B-cell epitopes in these proteins and selected those epitopes with sequences that were divergent in the corresponding orthologs in Homo sapiens, in Canis familiaris, and in Trypanosoma cruzi. We compared the performance of these peptides with the recombinant protein using ELISA. Both MAPK3 and MAPK4 recombinant proteins showed better specificity in the immunodiagnosis of human and canine leishmaniasis than soluble parasite antigens and the EIE-leishmaniose-visceral-canina-bio-manguinhos (EIE-LVC) kit. Furthermore, the performance of this serodiagnosis assay was improved using synthetic peptides corresponding to B-cell epitopes derived from both proteins.

  6. Effect of T-cell-epitope matching at HLA-DPB1 in recipients of unrelated-donor haemopoietic-cell transplantation: a retrospective study

    Science.gov (United States)

    Fleischhauer, Katharina; Gooley, Theodore; Malkki, Mari; Bardy, Peter; Bignon, Jean-Denis; Dubois, Valérie; Horowitz, Mary M; Madrigal, J Alejandro; Morishima, Yasuo; Oudshoorn, Machteld; Ringden, Olle; Spellman, Stephen; Velardi, Andrea; Zino, Elisabetta; Petersdorf, Effie W

    2013-01-01

    Summary Background The risks after unrelated-donor haemopoietic-cell transplantation with matched HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 alleles between donor and recipient (10/10 matched) can be decreased by selection of unrelated donors who also match for HLA-DPB1; however, such donors are difficult to find. Classification of HLA-DPB1 mismatches based on T-cell-epitope groups could identify mismatches that might be tolerated (permissive) and those that would increase risks (non-permissive) after transplantation. We did a retrospective study to compare outcomes between permissive and non-permissive HLA-DPB1 mismatches in unrelated-donor haemopoietic-cell transplantation. Methods HLA and clinical data for unrelated-donor transplantations submitted to the International Histocompatibility Working Group in haemopoietic-cell transplantation were analysed retrospectively. HLA-DPB1 T-cell-epitope groups were assigned according to a functional algorithm based on alloreactive T-cell crossreactivity patterns. Recipients and unrelated donors matching status were classified as HLA-DPB1 match, non-permissive HLA-DPB1 mismatch (those with mismatched T-cell-epitope groups), or permissive HLA-DPB1 mismatch (those with matched T-cell-epitope groups). The clinical outcomes assessed were overall mortality, non-relapse mortality, relapse, and severe (grade 3–4) acute graft-versus-host disease (aGvHD). Findings Of 8539 transplantations, 5428 (64%) were matched for ten of ten HLA alleles (HLA 10/10 matched) and 3111 (36%) for nine of ten alleles (HLA 9/10 matched). Of the group overall, 1719 (20%) were HLA-DPB1 matches, 2670 (31%) non-permissive HLA-DPB1 mismatches, and 4150 (49%) permissive HLA-DPB1 mismatches. In HLA 10/10-matched transplantations, non-permissive mismatches were associated with a significantly increased risk of overall mortality (hazard ratio [HR] 1·15, 95% CI 1·05–1·25; p=0·002), non-relapse mortality (1·28, 1·14–1·42; pHLA-DPB1 mismatches and HLA-DPB1

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-02-02

    Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Discharge cell for ozone generator

    Science.gov (United States)

    Nakatsuka, Suguru

    2000-01-01

    A discharge cell for use in an ozone generator is provided which can suppress a time-related reduction in ozone concentration without adding a catalytic gas such as nitrogen gas to oxygen gas as a raw material gas. The discharge cell includes a pair of electrodes disposed in an opposed spaced relation with a discharge space therebetween, and a dielectric layer of a three-layer structure consisting of three ceramic dielectric layers successively stacked on at least one of the electrodes, wherein a first dielectric layer of the dielectric layer contacting the one electrode contains no titanium dioxide, wherein a second dielectric layer of the dielectric layer exposed to the discharge space contains titanium dioxide in a metal element ratio of not lower than 10 wt %.

  10. Identification of Murine B-Cell and T-Cell Epitopes of Escherichia coli Outer Membrane Protein F with Synthetic Polypeptides

    Science.gov (United States)

    Williams, Kristina M.; Bigley, Elmer C.; Raybourne, Richard B.

    2000-01-01

    The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on

  11. Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production.

    Science.gov (United States)

    Oyarzún, Patricio; Kobe, Bostjan

    2016-03-03

    Novel vaccination approaches based on rational design of B- and T-cell epitopes - epitope-based vaccines - are making progress in the clinical trial pipeline. The epitope-focused recombinant protein-based malaria vaccine (termed RTS,S) is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease. Progress made on methods such as recombinant DNA technology, advanced cell-culture techniques, immunoinformatics and rational design of immunogens are driving the development of these novel concepts. Synthetic recombinant proteins comprising both B- and T-cell epitopes can be efficiently produced through modern biotechnology and bioprocessing methods, and can enable the induction of large repertoires of immune specificities. In particular, the inclusion of appropriate CD4+ T-cell epitopes is increasingly considered a key vaccine component to elicit robust immune responses, as suggested by results coming from HIV-1 clinical trials. In silico strategies for vaccine design are under active development to address genetic variation in pathogens and several broadly protective "universal" influenza and HIV-1 vaccines are currently at different stages of clinical trials. Other methods focus on improving population coverage in target populations by rationally considering specificity and prevalence of the HLA proteins, though a proof-of-concept in humans has not been demonstrated yet. Overall, we expect immunoinformatics and bioprocessing methods to become a central part of the next-generation epitope-based vaccine development and production process.

  12. Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation.

    Directory of Open Access Journals (Sweden)

    Angelo Duarte

    Full Text Available Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

  13. Cumulative autoimmunity: T cell clones recognizing several self-epitopes exhibit enhanced pathogenicity

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    Roland S. LIBLAU

    2011-10-01

    Full Text Available T cell receptor (TCR recognition is intrinsically polyspecific. In the field of autoimmunity, recognition of both self- and microbial peptides by a single TCR has led to the concept of molecular mimicry. However, findings made by our group and others clearly demonstrate that a given TCR can also recognize multiple distinct self-peptides. Based on our data we postulate that recognition of several self-peptides is an important parameter governing the pathogenicity of an autoreactive T cell, and refer to this function as ‘cumulative autoimmunity’. The mechanisms of such increased pathogenicity, and the implications of cumulative autoimmunity regarding the pathophysiology of T cell-mediated autoimmune diseases will be discussed.

  14. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N SARS-CoV protein using a phage display approach

    Directory of Open Access Journals (Sweden)

    Grasso Felicia

    2005-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

  15. Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach

    Science.gov (United States)

    Flego, Michela; Di Bonito, Paola; Ascione, Alessandro; Zamboni, Silvia; Carattoli, Alessandra; Grasso, Felicia; Cassone, Antonio; Cianfriglia, Maurizio

    2005-01-01

    Background Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease. Methods The human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry. Results Human scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells. Conclusion The human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells. PMID:16171519

  16. Elimination of immunodominant epitopes from multispecific DNA-based vaccines allows induction of CD8 T cells that have a striking antiviral potential

    DEFF Research Database (Denmark)

    Riedl, Petra; Wieland, Andreas; Lamberth, Kasper

    2009-01-01

    Immunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV...... cell immunity by multidomain Ags. The "weak" (i.e., easily suppressed) K(b)/C(93-100)-specific CD8 T cell response was efficiently elicited by a HBV core Ag-encoding vector in 1.4HBV-S(mut) tg mice (that harbor a replicating HBV genome that produces HBV surface, core, and precore Ag in the liver). K......(b)/C(93-100)-specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-S(mut) transgenic mice where they suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific antiviral effects to HBV...

  17. Mutational Analysis of Gene Fusions Predicts Novel MHC Class I-Restricted T-Cell Epitopes and Immune Signatures in a Subset of Prostate Cancer.

    Science.gov (United States)

    Kalina, Jennifer L; Neilson, David S; Lin, Yen-Yi; Hamilton, Phineas T; Comber, Alexandra P; Loy, Emma M H; Sahinalp, S Cenk; Collins, Colin C; Hach, Faraz; Lum, Julian J

    2017-12-15

    Purpose: Gene fusions are frequently found in prostate cancer and may result in the formation of unique chimeric amino acid sequences (CASQ) that span the breakpoint of two fused gene products. This study evaluated the potential for fusion-derived CASQs to be a source of tumor neoepitopes, and determined their relationship to patterns of immune signatures in prostate cancer patients. Experimental Design: A computational strategy was used to identify CASQs and their corresponding predicted MHC class I epitopes using RNA-Seq data from The Cancer Genome Atlas of prostate tumors. In vitro peptide-specific T-cell expansion was performed to identify CASQ-reactive T cells. A multivariate analysis was used to relate patterns of in silico -predicted tumor-infiltrating immune cells with prostate tumors harboring these mutational events. Results: Eighty-seven percent of tumors contained gene fusions with a mean of 12 per tumor. In total, 41% of fusion-positive tumors were found to encode CASQs. Within these tumors, 87% gave rise to predicted MHC class I-binding epitopes. This observation was more prominent when patients were stratified into low- and intermediate/high-risk categories. One of the identified CASQ from the recurrent TMPRSS2:ERG type VI fusion contained several high-affinity HLA-restricted epitopes. These peptides bound HLA-A*02:01 in vitro and were recognized by CD8 + T cells. Finally, the presence of fusions and CASQs were associated with expression of immune cell infiltration. Conclusions: Mutanome analysis of gene fusion-derived CASQs can give rise to patient-specific predicted neoepitopes. Moreover, these fusions predicted patterns of immune cell infiltration within a subgroup of prostate cancer patients. Clin Cancer Res; 23(24); 7596-607. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

    Science.gov (United States)

    Dubois, Louise; Stridsberg, Mats; Kharaziha, Pedram; Chioureas, Dimitris; Meersman, Niels; Panaretakis, Theocharis; Ronquist, K Göran

    2015-07-01

    Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers. © 2015 Wiley Periodicals, Inc.

  19. Autophagy creates a CTL epitope that mimics tumor-associated antigens.

    Directory of Open Access Journals (Sweden)

    Ayako Demachi-Okamura

    Full Text Available The detailed mechanisms responsible for processing tumor-associated antigens and presenting them to CTLs remain to be fully elucidated. In this study, we demonstrate a unique CTL epitope generated from the ubiquitous protein puromycin-sensitive aminopeptidase, which is presented via HLA-A24 on leukemic and pancreatic cancer cells but not on normal fibroblasts or EBV-transformed B lymphoblastoid cells. The generation of this epitope requires proteasomal digestion and transportation from the endoplasmic reticulum to the Golgi apparatus and is sensitive to chloroquine-induced inhibition of acidification inside the endosome/lysosome. Epitope liberation depends on constitutively active autophagy, as confirmed with immunocytochemistry for the autophagosome marker LC3 as well as RNA interference targeting two different autophagy-related genes. Therefore, ubiquitously expressed proteins may be sources of specific tumor-associated antigens when processed through a unique mechanism involving autophagy.

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

    Science.gov (United States)

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

    2010-07-19

    Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination. (c) 2010 Elsevier Ltd. All rights reserved.

  1. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

    Science.gov (United States)

    Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio

    2015-01-01

    The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis. We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis. The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

  2. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

    Directory of Open Access Journals (Sweden)

    Daniel Menezes-Souza

    2015-01-01

    Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

  3. Epitope discovery with phylogenetic hidden Markov models.

    LENUS (Irish Health Repository)

    Lacerda, Miguel

    2010-05-01

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

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

    DEFF Research Database (Denmark)

    Adorini, L; Sette, A; Buus, S

    1988-01-01

    but not the binding to I-Ed molecules, whereas, as shown by binding data and competition experiments, an Arg----His substitution at position 114 profoundly impairs the capacity of the peptide to interact with I-Ed molecules. In agreement with these results, [Lys113]HEL-(105-120)-peptide but not [His114]HEL-(105......-120)-peptide binds to I-Ed but not to I-Ad molecules. Conservative or semiconservative substitutions at positions 113 (Asn----Lys), 114 (Arg----His), or 115 (Cys----Ala) abrogate the ability of HEL-(105-120) to activate T cells. Substitutions at residues 113 and 115 affect T-cell recognition...

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

    Directory of Open Access Journals (Sweden)

    Jairo Andres Fonseca

    Full Text Available 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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Christina Gerstner

    2016-11-01

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

  8. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Directory of Open Access Journals (Sweden)

    Jonathan D Steckbeck

    2010-12-01

    Full Text Available The C-terminal tail (CTT of the HIV-1 gp41 envelope (Env protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs. Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  9. Malondialdehyde epitopes as mediators of sterile inflammation.

    Science.gov (United States)

    Busch, Clara J; Binder, Christoph J

    2017-04-01

    Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products

    NARCIS (Netherlands)

    Platteel, Anouk C M; Mishto, Michele; Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Busch, Dirk H; Kloetzel, Peter M; Sijts, Alice J A M

    CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed

  11. Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project."

    DEFF Research Database (Denmark)

    Lauemøller, S L; Kesmir, C; Corbet, S L

    2000-01-01

    Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate discrimin......Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate...... discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles...

  12. Identification and characterization of B-cell epitopes in the DBL4e domain of VAR2CSA

    DEFF Research Database (Denmark)

    Ditlev, Sisse B; Nielsen, Morten A; Resende, Mafalda

    2012-01-01

    capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope...

  13. Characterization of murine B-cell epitopes on the Mycobacterium leprae proline-rich antigen by use of synthetic peptides

    NARCIS (Netherlands)

    Klatser, P. R.; de Wit, M. Y.; Kolk, A. H.; Hartskeerl, R. A.

    1991-01-01

    Using synthetic peptides representing overlapping sequences of the 100-amino-acid-long N-terminal region of the proline-rich antigen of Mycobacterium leprae (PRA), we have mapped the epitopes in the primary structure of PRA recognized by four monoclonal antibodies. The M. leprae-specific monoclonal

  14. Interdisciplinary Analysis of HIV-Specific CD8(+) T Cell Responses against Variant Epitopes Reveals Restricted TCR Promiscuity

    DEFF Research Database (Denmark)

    Hoof, Ilka; Perez, C.L.; Buggert, M.

    2010-01-01

    HIV-1 specific CTL responses play a key role in limiting viral replication. CTL responses are sensitive to viral escape mutations, which influence recognition of the virus. Although CTLs have been shown to recognize epitope variants, the extent of this cross-reactivity has not been quantitatively...

  15. Decellularization of bovine anterior cruciate ligament tissues minimizes immunogenic reactions to alpha-gal epitopes by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Yoshida, Ryu; Vavken, Patrick; Murray, Martha M

    2012-10-01

    Rupture of ACL is a common injury. While the current surgical treatments are effective, many patients still suffer from precocious osteoarthritis, and there is an increasing interest in bioengineering approaches to improve ACL repair. Bovine collagen is a material currently in use for tissue engineering of ligaments. The alpha-gal epitopes found on bovine cells are a source of immunogenic stimulus for human cells. In this study, we wished to determine if those epitopes could be removed sufficiently to mitigate an immunogenic response using either a decellularization protocol or decellularization followed by alpha-galactosidase treatment. Bovine ACLs were treated with Triton-X, sodium deoxycholate, ribonuclease, and deoxyribonuclease to remove cells. A subset of the decellularized tissues was further treated with alpha-galactosidase. Human peripheral blood mononuclear cells (PBMCs) were exposed to untreated, decellularized, and alpha-galactosidase-treated tissues, and PBMC migration and IL-6 release were measured. PBMCs were significantly more attracted to untreated ACL compared to decellularized or alpha-galactosidase-treated tissue, but no difference was seen between the two treatment groups. PBMCs also released significantly more IL-6 when exposed to untreated tissue compared to decellularized ACL or alpha-galactosidase-treated ACL, but no difference was seen between the two treatment groups. Immunohistochemistry using anti-alpha-gal antibody detected the epitopes throughout the untreated ACL, but similar areas of reaction were not seen on decellularized or alpha-galactosidase-treated ACL. These results suggest that our decellularization protocol minimizes the immunogenic reactions of human PBMCs to bovine ACL tissue. Therefore, decellularized bovine ACL tissue may be a safe, effective biomaterial for ACL injury treatments. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Role of peptide processing predictions in T cell epitope identification: contribution of different prediction programs.

    Science.gov (United States)

    Calis, Jorg J A; Reinink, Peter; Keller, Christin; Kloetzel, Peter M; Keşmir, Can

    2015-02-01

    Proteolysis is the general term to describe the process of protein degradation into peptides. Proteasomes are the main actors in cellular proteolysis, and their activity can be measured in in vitro digestion experiments. However, in vivo proteolysis can be different than what is measured in these experiments if other proteases participate or if proteasomal activity is different in vivo. The in vivo proteolysis can be measured only indirectly, by the analysis of peptides presented on MHC-I molecules. MHC-I presented peptides are protected from further degradation, thus enabling an indirect view on the underlying in vivo proteolysis. The ligands presented on different MHC-I molecules enable different views on this process; in combination, they might give a complete picture. Based on in vitro proteasome-only digestions and MHC-I ligand data, different proteolysis predictors have been developed. With new in vitro digestion and MHC-I ligand data sets, we benchmarked how well these predictors capture in vitro proteasome-only activity and in vivo whole-cell proteolysis, respectively. Even though the in vitro proteasome digestion patterns were best captured by methods trained on such data (ProteaSMM and NetChop 20S), the in vivo whole-cell proteolysis was best predicted by a method trained on MHC-I ligand data (NetChop Cterm). Follow-up analysis showed that the likely source of this difference is the activity from proteases other than the proteasome, such as TPPII. This non-proteasomal in vivo activity is captured by NetChop Cterm and should be taken into account in MHC-I ligand predictions.

  17. Exposure to Melan-A/MART-126-35 tumor epitope specific CD8(+)T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS).

    Science.gov (United States)

    Ebstein, Frédéric; Keller, Martin; Paschen, Annette; Walden, Peter; Seeger, Michael; Bürger, Elke; Krüger, Elke; Schadendorf, Dirk; Kloetzel, Peter-M; Seifert, Ulrike

    2016-05-04

    Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

  18. Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines.

    Science.gov (United States)

    Roukaerts, Inge D M; Grant, Chris K; Theuns, Sebastiaan; Christiaens, Isaura; Acar, Delphine D; Van Bockstael, Sebastiaan; Desmarets, Lowiese M B; Nauwynck, Hans J

    2017-01-02

    Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission. Copyright © 2016. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2013-09-01

    Hepsin is a type II transmembrane serine protease that is overexpressed in prostate cancer, and it is associated with prostate cancer cellular migration and invasion. Therefore, HPN is a biomarker for prostate cancer. CD8(+) T cells play an important role in tumour immunity. This study predicted and identified HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in human hepsin protein. HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: (1) a computer program generated predicted epitopes from the amino acid sequence of human hepsin; (2) an HLA-A2-binding assay detected the affinity of the predicted epitopes to the HLA-A2 molecule; (3) the primary T cell response against the predicted epitopes was stimulated in vitro; and (4) the induced CTLs towards different types of hepsin- or HLA-A2-expressing prostate cancer cells were detected. Five candidate peptides were identified. The effectors that were induced by human hepsin epitopes containing residues 229 to 237 (Hpn229; GLQLGVQAV), 268 to 276 (Hpn268; PLTEYIQPV) and 191 to 199 (Hpn199; SLLSGDWVL) effectively lysed LNCaP prostate cancer cells that were hepsin-positive and HLA-A2 matched. These peptide-specific CTLs did not lyse normal liver cells with low hepsin levels. Hpn229, Hpn268 and Hpn199 increased the frequency of IFN-γ-producing T cells compared with the negative peptide. These results suggest that the Hpn229, Hpn268 and Hpn199 epitopes are novel HLA-A2-restricted CTL epitopes that are capable of inducing hepsin-specific CTLs in vitro. Hpn229, Hpn268 and Hpn199 peptide-based vaccines may be useful for immunotherapy in patients with prostate cancer. © 2013 John Wiley & Sons Ltd.

  20. BIITE: A Tool to Determine HLA Class II Epitopes from T Cell ELISpot Data.

    Directory of Open Access Journals (Sweden)

    Lies Boelen

    2016-03-01

    Full Text Available Activation of CD4+ T cells requires the recognition of peptides that are presented by HLA class II molecules and can be assessed experimentally using the ELISpot assay. However, even given an individual's HLA class II genotype, identifying which class II molecule is responsible for a positive ELISpot response to a given peptide is not trivial. The two main difficulties are the number of HLA class II molecules that can potentially be formed in a single individual (3-14 and the lack of clear peptide binding motifs for class II molecules. Here, we present a Bayesian framework to interpret ELISpot data (BIITE: Bayesian Immunogenicity Inference Tool for ELISpot; specifically BIITE identifies which HLA-II:peptide combination(s are immunogenic based on cohort ELISpot data. We apply BIITE to two ELISpot datasets and explore the expected performance using simulations. We show this method can reach high accuracies, depending on the cohort size and the success rate of the ELISpot assay within the cohort.

  1. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  2. Identification of an HLA-A2-restricted CD147 epitope that can induce specific CTL cytotoxicity against drug resistant MCF-7/Adr cells.

    Science.gov (United States)

    Qu, Chuang; Gao, Shuhui; Shao, Hongwei; Zhang, Wenfeng; Bo, Huabben; Lu, Xin; Chen, Tianjiao; Kou, Jing; Wang, Yue; Chen, Gui Si; Huang, Shulin; Shen, Han

    2018-04-01

    Cluster of differentiation (CD)147 is highly expressed in drug-resistant tumor cell lines and is involved in the formation of tumor drug resistance. Therefore, immunotherapy utilizing CD147 epitope peptides is a promising approach for the elimination of drug-resistant tumor cells. However, like most tumor-associated antigens (TAAs), CD147 belongs to the autoantigen category, and T cells that recognize high affinity, immunodominant epitopes from autoantigens are deleted though thymic negative selection. Furthermore, wild-type autoantigen peptides cannot effectively activate and expand T lymphocytes with lower affinity T cell receptors in vivo . However, mutations of TAA peptides have been demonstrated to increase the affinity of major histocompatibility complex molecules and their binding to T cell receptor molecules, leading to activation of T lymphocytes in vitro . In the present study, a high-affinity point mutation peptide, CD147 126-134 L2, was predicted by the human leukocyte antigen (HLA) binding prediction algorithm and its affinity was testified using a T2 binding assay. In addition, when peptide-specific cytotoxic T lymphocytes (CTLs) were stimulated with dendritic cells loaded with the CD147 126-134 L2 peptide under HLA-A*02:01 restriction, interferon-γ release and cytotoxicity assays showed that peptide-specific CTLs effectively cross-recognized and lysed T2 target cells loaded either with the wild-type (CD147 126-134 ) or mutated peptide (CD147 126-134 L2). Moreover, the CD147 126-134 L2 peptide-specific CTLs exerted strong cytotoxic activity against drug-resistant MCF-7/Adr cells, which express a high level of CD147 and are HLA-A*02:01-positive, but not against normal MCF-7 cells. Thus, this suggests that the wild-type peptide (CD147 126-134 ) is naturally presented on HLA-A*02:01 of CD147-expressing MCF-7/Adr cells and is cross-recognized by CTLs. In conclusion, an HLA-A*02:01-restricted CD147-point mutant epitope peptide was identified that induces

  3. Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested.

    Science.gov (United States)

    Leoff, Christine; Saile, Elke; Rauvolfova, Jana; Quinn, Conrad P; Hoffmaster, Alex R; Zhong, Wei; Mehta, Alok S; Boons, Geert-Jan; Carlson, Russell W; Kannenberg, Elmar L

    2009-06-01

    The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.

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

    Science.gov (United States)

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

    2017-09-29

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

  5. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

    OpenAIRE

    Boegel, Sebastian; L?wer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    2014-01-01

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel?opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antige...

  6. First quantitative assay of alpha-Gal in soft tissues: presence and distribution of the epitope before and after cell removal from xenogeneic heart valves.

    Science.gov (United States)

    Naso, F; Gandaglia, A; Iop, L; Spina, M; Gerosa, G

    2011-04-01

    Decellularized xenograft heart valves might be the ideal scaffolds for tissue engineered heart valves as the alternative to the currently used biological and mechanical prostheses. However, removal of the alpha-Gal epitope is a prerequisite to avoid hyperacute rejection of untreated xenograft material. The aim of this study was to develop an ELISA soft-tissue assay for alpha-Gal quantification in xenograft heart valves before and after a detergent-based (TriCol) or equivalent cell removal procedure. Leaflets from porcine valves were enzymatically digested to expose the epitope and reacted with the alpha-Gal monoclonal antibody M86 for its recognition. Rabbit erythrocytes were used as a reference for the quantification of alpha-Gal. Native aortic and pulmonary leaflets exhibited different epitope concentration: 4.33×10(11) vs. 7.12×10(11)/10 mg wet tissue (pvalves revealed a different alpha-Gal distribution within and among different leaflets. The pattern was consistent with immunofluorescence analysis and was unrelated to microvessel density distribution. After TriCol treatment alpha-Gal was no longer detectable in both pulmonary and aortic decellularized valves, confirming the ability of this method to remove both cells and alpha-Gal antigen. These results hold promise for a reliable quantitative evaluation of alpha-Gal in decellularized valves obtained from xenograft material for tissues engineering purposes. Additionally, this method is applicable to further evaluate currently used xenograft bioprostheses. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3).

    OpenAIRE

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

    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. Met...

  8. Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer

    Directory of Open Access Journals (Sweden)

    Heemskerk Mirjam HM

    2011-09-01

    Full Text Available Abstract Background Infection with high risk Human Papilloma Virus (HPV is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. Methods HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. Results Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. Conclusion Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

  9. Dominant epitopes and allergic cross-reactivity

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ipsen, H

    2000-01-01

    The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces...

  10. Epitope mapping of Brugia malayi ALT-2 and the development of a multi-epitope vaccine for lymphatic filariasis.

    Science.gov (United States)

    Madhumathi, J; Prince, P R; Rao, D N; Karande, A A; Reddy, M V R; Kaliraj, P

    2017-01-01

    Human lymphatic filariasis is a neglected tropical disease, causing permanent and long-term disability with severe immunopathology. Abundant larval transcript (ALT) plays a crucial role in parasite establishment in the host, due to its multi-faceted ability in host immune regulation. Although ALT protein is a key filarial target, its exact function is yet to be explored. Here, we report epitope mapping and a structural model of Brugia malayi ALT-2, leading to development of a multi-epitope vaccine. Structural analysis revealed that ALT represents unique parasitic defence proteins belonging to a toxin family that carries a 'knottin' fold. ALT-2 has been a favourite vaccine antigen and was protective in filarial models. Due to the immunological significance of ALT-2, we mapped B-cell epitopes systematically and identified two epitope clusters, 1-30 and 89-128. To explore the prophylactic potential of epitope clusters, a recombinant multi-epitopic gene comprising the epitopic domains was engineered and the protective efficacy of recombinant ALT epitope protein (AEP) was tested in the permissive model, Mastomys coucha. AEP elicited potent antibody responses with predominant IgG1 isotype and conferred significantly high protection (74.59%) compared to ALT-2 (61.95%). This proved that these epitopic domains are responsible for the protective efficacy of ALT-2 and engineering protective epitopes as a multi-epitope protein may be a novel vaccine strategy for complex parasitic infections.

  11. Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections

    Directory of Open Access Journals (Sweden)

    Liu Wen-Xin

    2010-09-01

    Full Text Available Abstract Background Differential diagnose of Japanese encephalitis virus (JEV infection from other flavivirus especially West Nile virus (WNV and Dengue virus (DV infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. Results To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120 was detected by enzyme-linked immunosorbent assay (ELISA. By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118. This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. Conclusion Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

  12. Primordial germ cells in the dorsal mesentery of the chicken embryo demonstrate left-right asymmetry and polarized distribution of the EMA1 epitope.

    Science.gov (United States)

    Hen, Gideon; Friedman-Einat, Miriam; Sela-Donenfeld, Dalit

    2014-05-01

    Despite the importance of the chicken as a model system, our understanding of the development of chicken primordial germ cells (PGCs) is far from complete. Here we characterized the morphology of PGCs at different developmental stages, their migration pattern in the dorsal mesentery of the chicken embryo, and the distribution of the EMA1 epitope on PGCs. The spatial distribution of PGCs during their migration was characterized by immunofluorescence on whole-mounted chicken embryos and on paraffin sections, using EMA1 and chicken vasa homolog antibodies. While in the germinal crescent PGCs were rounded and only 25% of them were labeled by EMA1, often seen as a concentrated cluster on the cell surface, following extravasation and migration in the dorsal mesentery PGCs acquired an elongated morphology, and 90% exhibited EMA1 epitope, which was concentrated at the tip of the pseudopodia, at the contact sites between neighboring PGCs. Examination of PGC migration in the dorsal mesentery of Hamburger and Hamilton stage 20-22 embryos demonstrated a left-right asymmetry, as migration of cells toward the genital ridges was usually restricted to the right, rather than the left, side of the mesentery. Moreover, an examination of another group of cells that migrate through the dorsal mesentery, the enteric neural crest cells, revealed a similar preference for the right side of the mesentery, suggesting that the migratory pathway of PGCs is dictated by the mesentery itself. Our findings provide new insights into the migration pathway of PGCs in the dorsal mesentery, and suggest a link between EMA1, PGC migration and cell-cell interactions. These findings may contribute to a better understanding of the mechanism underlying migration of PGCs in avians. © 2014 Anatomical Society.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding...... for predicting peptides binding to specific MHC class I alleles. The method combines advanced automatic scoring matrix generation with empirical position specific differential anchor weighting. The method leads to predictions with a comparable or higher accuracy than other established prediction servers, even...

  14. Peptide-Based Vaccinology: Experimental and Computational Approaches to Target Hypervariable Viruses through the Fine Characterization of Protective Epitopes Recognized by Monoclonal Antibodies and the Identification of T-Cell-Activating Peptides

    Directory of Open Access Journals (Sweden)

    Matteo Castelli

    2013-01-01

    Full Text Available Defining immunogenic domains of viral proteins capable of eliciting a protective immune response is crucial in the development of novel epitope-based prophylactic strategies. This is particularly important for the selective targeting of conserved regions shared among hypervariable viruses. Studying postinfection and postimmunization sera, as well as cloning and characterization of monoclonal antibodies (mAbs, still represents the best approach to identify protective epitopes. In particular, a protective mAb directed against conserved regions can play a key role in immunogen design and in human therapy as well. Experimental approaches aiming to characterize protective mAb epitopes or to identify T-cell-activating peptides are often burdened by technical limitations and can require long time to be correctly addressed. Thus, in the last decade many epitope predictive algorithms have been developed. These algorithms are continually evolving, and their use to address the empirical research is widely increasing. Here, we review several strategies based on experimental techniques alone or addressed by in silico analysis that are frequently used to predict immunogens to be included in novel epitope-based vaccine approaches. We will list the main strategies aiming to design a new vaccine preparation conferring the protection of a neutralizing mAb combined with an effective cell-mediated response.

  15. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines.

    Science.gov (United States)

    Boegel, Sebastian; Löwer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel-opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been distributed in many publications. Here, we determine the 4-digit HLA type and HLA expression of 167 cancer and 10 non-cancer cell lines from publically available RNA-Seq data. We use standard NGS RNA-Seq short reads from "whole transcriptome" sequencing, map reads to known HLA types, and statistically determine HLA type, heterozygosity, and expression. First, we present previously unreported HLA Class I and II genotypes. Second, we determine HLA expression levels in each cancer cell line, providing insights into HLA downregulation and loss in cancer. Third, using these results, we provide a fundamental cell line "barcode" to track samples and prevent sample annotation swaps and contamination. Fourth, we integrate the cancer cell-line specific HLA types and HLA expression with available cell-line specific mutation information and existing HLA binding prediction algorithms to make a catalog of predicted antigenic mutations in each cell line. The compilation of our results are a fundamental resource for all researchers selecting specific cancer cell lines based on the HLA type and HLA expression, as well as for the development of immunotherapeutic tools for novel cancer treatment modalities.

  16. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

    Science.gov (United States)

    Boegel, Sebastian; Löwer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    2014-01-01

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel­opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been distributed in many publications. Here, we determine the 4-digit HLA type and HLA expression of 167 cancer and 10 non-cancer cell lines from publically available RNA-Seq data. We use standard NGS RNA-Seq short reads from “whole transcriptome” sequencing, map reads to known HLA types, and statistically determine HLA type, heterozygosity, and expression. First, we present previously unreported HLA Class I and II genotypes. Second, we determine HLA expression levels in each cancer cell line, providing insights into HLA downregulation and loss in cancer. Third, using these results, we provide a fundamental cell line “barcode” to track samples and prevent sample annotation swaps and contamination. Fourth, we integrate the cancer cell-line specific HLA types and HLA expression with available cell-line specific mutation information and existing HLA binding prediction algorithms to make a catalog of predicted antigenic mutations in each cell line. The compilation of our results are a fundamental resource for all researchers selecting specific cancer cell lines based on the HLA type and HLA expression, as well as for the development of immunotherapeutic tools for novel cancer treatment modalities. PMID:25960936

  17. Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

    Science.gov (United States)

    Szabados, Hajnalka; Uray, Katalin; Majer, Zsuzsa; Silló, Pálma; Kárpáti, Sarolta; Hudecz, Ferenc; Bősze, Szilvia

    2015-09-01

    Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  18. Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis.

    Science.gov (United States)

    Bastos, Luciana M; Macêdo, Arlindo G; Silva, Murilo V; Santiago, Fernanda M; Ramos, Eliezer L P; Santos, Fabiana A A; Pirovani, Carlos P; Goulart, Luiz R; Mineo, Tiago W P; Mineo, José R

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Luciana Machado Bastos

    2016-06-01

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

  20. Identification of cross-reacting T-cell epitopes in structural and non-structural proteins of swine and pandemic H1N1 influenza A virus strains in pigs

    DEFF Research Database (Denmark)

    Baratelli, Massimiliano; Pedersen, Lasse Eggers; Trebbien, Ramona

    2017-01-01

    , reverse vaccinology was applied to identify cross-reacting MHC class I T-cell epitopes from two different SwIV H1 lineages in pigs. In silico prediction followed by in vitro and in vivo testing was used to identify SLA-1*0702 T-cell epitopes in heterologous SwIV-infected pigs. Following viral infection......Heterologous protection against swine influenza viruses (SwIVs) of different lineages is an important concern for the pig industry. Cross-protection between 'avian-like' H1N1 and 2009 pandemic H1N1 lineages has been observed previously, indicating the involvement of cross-reacting T-cells. Here......, tetramer specific T-cell populations were identified. The majority of the identified T-cell epitopes were conserved between the examined lineages, suggesting that targeting cross-reactive T-cell epitopes could be used to improve vaccines against SwIV in SLA-1*0702-positive pigs....

  1. Fuel dissipater for pressurized fuel cell generators

    Science.gov (United States)

    Basel, Richard A.; King, John E.

    2003-11-04

    An apparatus and method are disclosed for eliminating the chemical energy of fuel remaining in a pressurized fuel cell generator (10) when the electrical power output of the fuel cell generator is terminated during transient operation, such as a shutdown; where, two electrically resistive elements (two of 28, 53, 54, 55) at least one of which is connected in parallel, in association with contactors (26, 57, 58, 59), a multi-point settable sensor relay (23) and a circuit breaker (24), are automatically connected across the fuel cell generator terminals (21, 22) at two or more contact points, in order to draw current, thereby depleting the fuel inventory in the generator.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  3. Difference in TB10.4 T-cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Billeskov, Rolf; Grandal, Michael V; Poulsen, Christian

    2010-01-01

    Most novel vaccines against infectious diseases are based on recombinant Ag; however, only few studies have compared Ag-specific immune responses induced by natural infection with that induced by the same Ag in a recombinant form. Here, we studied the epitope recognition pattern of the tuberculosis...... vaccine Ag, TB10.4, in a recombinant form, or when expressed by the pathogen Mycobacterium tuberculosis (M.tb), or by the current anti-tuberculosis vaccine, Mycobacterium bovis BCG. We showed that BCG and M.tb induced a similar CD4(+) T-cell specific TB10.4 epitope-pattern, which differed completely from...... that induced by recombinant TB10.4. This difference was not due to post-translational modifications of TB10.4 or because TB10.4 is secreted from BCG and M.tb as a complex with Rv0287. In addition, BCG and TB10.4/CAF01 were both taken up by DC and macrophages in vivo, and in vitro uptake experiments revealed...

  4. Antibody recognition of cathepsin L1-derived peptides in Fasciola hepatica-infected and/or vaccinated cattle and identification of protective linear B-cell epitopes.

    Science.gov (United States)

    Garza-Cuartero, Laura; Geurden, Thomas; Mahan, Suman M; Hardham, John M; Dalton, John P; Mulcahy, Grace

    2018-02-08

    Fasciola hepatica infection causes important economic losses in livestock and food industries around the world. In the Republic of Ireland F. hepatica infection has an 76% prevalence in cattle. Due to the increase of anti-helminthic resistance, a vaccine-based approach to control of Fasciolosis is urgently needed. A recombinant version of the cysteine protease cathepsin L1 (rmFhCL1) from F. hepatica has been a vaccine candidate for many years. We have found that vaccination of cattle with this immunodominant antigen has provided protection against infection in some experimental trials, but not in others. Differential epitope recognition between animals could be a source of variable levels of vaccine protection. Therefore, we have characterised for first time linear B-cell epitopes recognised within the FhCL1 protein using sera from F. hepatica-infected and/or vaccinated cattle from two independent trials. Results showed that all F. hepatica infected animals recognised the region 19-31 of FhCL1, which is situated in the N-terminal part of the pro-peptide. Vaccinated animals that showed fluke burden reduction elicited antibodies that bound to the regions 120-137, 145-155, 161-171 of FhCL1, which were not recognised by non-protected animals. This data, together with the high production of specific IgG2 in animals showing vaccine efficacy, suggest important targets for vaccine development. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified from herpes simplex virus glycoprotein D

    DEFF Research Database (Denmark)

    Chentoufi, Aziz Alami; Zhang, Xiuli; Lamberth, Kasper

    2008-01-01

    epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10...

  6. B Epitope Multiplicity and B/T Epitope Orientation Influence Immunogenicity of Foot-and-Mouth Disease Peptide Vaccines

    Directory of Open Access Journals (Sweden)

    Esther Blanco

    2013-01-01

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

  7. Proof of principle for epitope-focused vaccine design

    Science.gov (United States)

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

    2014-03-01

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

  8. Enhancing antibody patent protection using epitope mapping information

    Science.gov (United States)

    Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J.

    2018-01-01

    ABSTRACT As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents. PMID:29120697

  9. Enhancing antibody patent protection using epitope mapping information.

    Science.gov (United States)

    Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J

    As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents.

  10. In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response.

    Directory of Open Access Journals (Sweden)

    Negar Seyed

    2011-09-01

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

  11. Generation of functional eyes from pluripotent cells.

    Directory of Open Access Journals (Sweden)

    Andrea S Viczian

    2009-08-01

    Full Text Available Pluripotent cells such as embryonic stem (ES and induced pluripotent stem (iPS cells are the starting point from which to generate organ specific cell types. For example, converting pluripotent cells to retinal cells could provide an opportunity to treat retinal injuries and degenerations. In this study, we used an in vivo strategy to determine if functional retinas could be generated from a defined population of pluripotent Xenopus laevis cells. Animal pole cells isolated from blastula stage embryos are pluripotent. Untreated, these cells formed only epidermis, when transplanted to either the flank or eye field. In contrast, misexpression of seven transcription factors induced the formation of retinal cell types. Induced retinal cells were committed to a retinal lineage as they formed eyes when transplanted to the flanks of developing embryos. When the endogenous eye field was replaced with induced retinal cells, they formed eyes that were molecularly, anatomically, and electrophysiologically similar to normal eyes. Importantly, induced eyes could guide a vision-based behavior. These results suggest the fate of pluripotent cells may be purposely altered to generate multipotent retinal progenitor cells, which differentiate into functional retinal cell classes and form a neural circuitry sufficient for vision.

  12. Mucosal tolerance and suppression of collagen-induced arthritis (CIA) induced by nasal inhalation of synthetic peptide 184-198 of bovine type II collagen (CII) expressing a dominant T cell epitope

    Science.gov (United States)

    STAINES, N. A.; HARPER, N.; WARD, F. J.; MALMSTRÖM, V.; HOLMDAHL, R.; BANSAL, S.

    1996-01-01

    The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RT1u haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184-198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine-specific, but cross-reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence were also autoantigenic. Use of independently synthesized and purified 184-198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA; this was found to delay the onset of disease, reduce mean disease severity, shift the anti-CII antibody response to favour antibodies of the IgGl, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184-198 peptide. The dominant 184-198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre-treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen. PMID:8608633

  13. Analysis of spontaneous tumor-specific CD4 T-cell immunity in lung cancer using promiscuous HLA-DR telomerase-derived epitopes: potential synergistic effect with chemotherapy response.

    Science.gov (United States)

    Godet, Yann; Fabre, Elizabeth; Dosset, Magalie; Lamuraglia, Michele; Levionnois, Emeline; Ravel, Patrice; Benhamouda, Nadine; Cazes, Aurélie; Le Pimpec-Barthes, Françoise; Gaugler, Beatrice; Langlade-Demoyen, Pierre; Pivot, Xavier; Saas, Philippe; Maillère, Bernard; Tartour, Eric; Borg, Christophe; Adotévi, Olivier

    2012-05-15

    To investigate the presence and impact of spontaneous telomerase-specific CD4 T-cell responses in cancer patients. A multistep approach was used to design novel pan-HLA-DR-restricted peptides from telomerase. T-cell clones isolated from cancer patients were used to characterize the polarization of telomerase-specific CD4 response. The presence of spontaneous CD4 T-cell response against telomerase was monitored in 84 metastatic non-small cell lung cancer (NSCLC) patients before first-line chemotherapy (CT) using IFN-γ ELISPOT assay. Then we analyzed the impact of the pretherapeutic telomerase-specific CD4 T immunity on clinical outcome in patients according to their respective response to CT. We described four novel telomerase-derived CD4 epitopes referred as universal cancer peptides (UCP) that effectively bind to most commonly found human MHC class II alleles. UCP-specific CD4 T-cell repertoire is present in human and UCP-specific CD4 T-cell clones generated from cancer patients exhibited high avidity and are Th1 polarized. Significant frequency (38%) of naturally occurring UCP-specific T-cell responses were detected before CT in advanced NSCLC but not in healthy volunteers. This response was shown to significantly increase overall survival (OS) of patients responding to CT (Median OS: 53 vs. 40 weeks, P = 0.034). These results show for the first time a potential synergistic effect of telomerase-specific CD4 T-cell response with CT response in NSCLC and underline the potential role of tumor-specific CD4 T-cell response on the efficiency of conventional anticancer therapy. ©2012 AACR.

  14. A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M)

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; He, Y.; Veidal, S. S.

    2011-01-01

    A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL......) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung-and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically...

  15. Computational screening of Six Antigens for potential MHC class II restricted epitopes and evaluating its CD4+ T-Cell Responsiveness against Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Manas Ranjan

    2017-12-01

    Full Text Available Visceral leishmaniasis is one of the most neglected tropical diseases for which no vaccine exists. In spite of extensive efforts, no successful vaccine is available against this dreadful infectious disease. To support the vaccine development, immunoinformatics approach was applied to search for potential MHC-classII restricted epitopes that can activate the immune cells. Initially, a total of 37 epitopes derived from six, stage dependent over expressed antigens were predicted, which were presented by at least 26 diverse MHC class II alleles including: DRB10101, DRB10301, DRB10401, DRB10404, DRB10405, DRB10701, DRB10802, DRB10901, DRB11101, DRB11302, DRB11501, DRB30101, DRB40101, DRB50101, DPA10103-DPB10401, DPA10103-DPB10201, DPA10201-DPB10101, DPA10103-DPB10301_DPB10401, DPA10301-DPB10402, DPA10201-DPB105021, DQA10102-DQB10602, DQA10401-DQB10402, DQA10501-QB10201, DQA10501-DQB10301, DQA10301-DQB10302 and DQA10101-DQB10501. Based on the population coverage analysis and HLA cross presentation ability, six epitopes namely, FDLFLFSNGAVVWWG (P1, YPVYPFLASNAALLN (P2, VYPFLASNAALLNLI (P3, LALLIMLYALIATQF (P4, LIMLYALIATQFSDD (P5, IMLYALIATQFSDDA (P6 were selected for further analysis. Stimulation with synthetic peptide alone or as a cocktail triggered the intracellular IFN-γ production. Moreover, specific IgG class of antibodies was detected in the serum of active VL cases against P1, P4, P and P6 in order to evaluate peptide effect on humoral immune response. Additionally, most of the peptides, except P2, were found to be non-inducer of CD4+ IL-10 against both active VL as well as treated VL subjects. Peptide immunogenicity was validated in BALB/c mice immunized with cocktail of synthetic peptide emulsified in complete Freund’s adjuvant/incomplete Freund’s adjuvant. The immunized splenocytes induced strong spleen cell proliferation upon parasite re-stimulation. Furthermore, an increased IFN-γ, IL-12, IL-17 and IL-22 production augmented with

  16. Helper T cell epitope-mapping reveals MHC-peptide binding affinities that correlate with T helper cell responses to pneumococcal surface protein A.

    Directory of Open Access Journals (Sweden)

    Rajesh Singh

    2010-02-01

    Full Text Available Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC class II. We also characterized spleen- and cervical lymph node (CLN-derived helper T lymphocyte (HTL cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA(199-246 consistently caused the greatest IFN-gamma, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4(+ T cells isolated from S. pneumonia strain EF3030-challeged F(1 (B6xBALB/c mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA(199-246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.

  17. Dendritic cell vaccination induces cross-reactive cytotoxic T lymphocytes specific for wild-type and natural variant human immunodeficiency virus type 1 epitopes in HLA-A*0201/Kb transgenic mice.

    Science.gov (United States)

    Abdel-Motal, U M; Friedline, R; Poligone, B; Pogue-Caley, R R; Frelinger, J A; Tisch, R

    2001-10-01

    Dendritic cells (DC) are highly efficient at inducing primary T cell responses. Consequently, DC are being investigated for their potential to prevent and/or treat human immunodeficiency virus type 1 (HIV-1) infection. In the current study, we examined the capacity of DC to elicit CD8+ cytotoxic T lymphocyte (CTL) reactivity against an HLA-A*0201-restricted HIV-1 reverse transcriptase (pol) epitope (residues 476-484) and two naturally occurring variants. Previous work demonstrated that the wild-type pol epitope is recognized by CTLs from HIV-1-infected individuals, whereas the variant pol epitopes are not, despite binding to HLA-A*0201. In agreement with these observations, parenteral administration of wild-type pol peptide induced HLA-A*0201-restricted CTL activity in A2Kb transgenic mice. In contrast, similar treatment with the two variant pol peptides failed to stimulate CTL reactivity, and this lack of immunogenicity correlated with reduced peptide:HLA-A*0201 complex stability. However, CTL responses were induced in A2Kb transgenic mice upon adoptive transfer of syngeneic bone marrow DC pulsed with the variant pol peptides. Furthermore, DC pulsed with the wild-type pol peptide elicited CTLs that cross-reacted with the variant pol epitopes. These results demonstrate that DC effectively expand the T cell repertoire of a given epitope to include cross-reactive T cell clonotypes. Accordingly, DC vaccination may aid in immune recognition of HIV-1 escape variants by broadening the T cell response. Copyright 2001 Academic Press.

  18. Pre-Vaccination Frequencies of Th17 Cells Correlate with Vaccine-Induced T-Cell Responses to Survivin-Derived Peptide Epitopes

    DEFF Research Database (Denmark)

    Køllgaard, Tania; Ugurel-Becker, Selma; Idorn, Manja

    2015-01-01

    Various subsets of immune regulatory cells are suggested to influence the outcome of therapeutic antigen-specific anti-tumor vaccinations. We performed an exploratory analysis of a possible correlation of pre-vaccination Th17 cells, MDSCs, and Tregs with both vaccination-induced T-cell responses......-generating study demonstrated that immune regulatory cells, in particular Th17 cells, play a relevant role for generation of the vaccine-induced anti-tumor immunity in cancer patients, hence warranting further investigation to test for validity as predictive biomarkers....... as well as clinical outcome in metastatic melanoma patients vaccinated with survivin-derived peptides. Notably, we observed dysfunctional Th1 and cytotoxic T cells, i.e. down-regulation of the CD3ζchain (p=0.001) and an impaired IFNγ-production (p=0.001) in patients compared to healthy donors, suggesting...

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

    Science.gov (United States)

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

    1998-04-01

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

  20. An epitope-specific DerG-PG70 LEAPS vaccine modulates T cell responses and suppresses arthritis progression in two related murine models of rheumatoid arthritis.

    Science.gov (United States)

    Mikecz, Katalin; Glant, Tibor T; Markovics, Adrienn; Rosenthal, Kenneth S; Kurko, Julia; Carambula, Roy E; Cress, Steve; Steiner, Harold L; Zimmerman, Daniel H

    2017-07-13

    Rheumatoid arthritis (RA) is an autoimmune joint disease maintained by aberrant immune responses involving CD4+ T helper (Th)1 and Th17 cells. In this study, we tested the therapeutic efficacy of Ligand Epitope Antigen Presentation System (LEAPS™) vaccines in two Th1 cell-driven mouse models of RA, cartilage proteoglycan (PG)-induced arthritis (PGIA) and PG G1-domain-induced arthritis (GIA). The immunodominant PG peptide PG70 was attached to a DerG or J immune cell binding peptide, and the DerG-PG70 and J-PG70 LEAPS vaccines were administered to the mice after the onset of PGIA or GIA symptoms. As indicated by significant decreases in visual and histopathological scores of arthritis, the DerG-PG70 vaccine inhibited disease progression in both PGIA and GIA, while the J-PG70 vaccine was ineffective. Splenic CD4+ cells from DerG-PG70-treated mice were diminished in Th1 and Th17 populations but enriched in Th2 and regulatory T (Treg) cells. In vitro spleen cell-secreted and serum cytokines from DerG-PG70-treated mice demonstrated a shift from a pro-inflammatory to an anti-inflammatory/regulatory profile. DerG-PG70 peptide tetramers preferentially bound to CD4+ T-cells of GIA spleen cells. We conclude that the DerG-PG70 vaccine (now designated CEL-4000) exerts its therapeutic effect by interacting with CD4+ cells, which results in an antigen-specific down-modulation of pathogenic T-cell responses in both the PGIA and GIA models of RA. Future studies will need to determine the potential of LEAPS vaccination to provide disease suppression in patients with RA. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    Science.gov (United States)

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

    2013-03-01

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

  2. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    Science.gov (United States)

    Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes

    2016-06-01

    Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics

  3. Field Effect Microparticle Generation for Cell Microencapsulation.

    Science.gov (United States)

    Hsu, Brend Ray-Sea; Fu, Shin-Huei

    2017-01-01

    The diameter and sphericity of alginate-poly-L-lysine-alginate microcapsules, determined by the size and the shape of calcium alginate microspheres, affect their in vivo durability and biocompatibility and the results of transplantation. The commonly used air-jet spray method generates microspheres with a wider variation in diameter, larger sphere morphology, and evenly distributed encapsulated cells. In order to overcome these drawbacks, we designed a field effect microparticle generator to create a stable electric field to prepare microparticles with a smaller diameter and more uniform morphology. Using this electric field microparticle generator the encapsulated cells will be located at the periphery of the microspheres, and thus the supply of oxygen and nutrients for the encapsulated cells will be improved compared with the centrally located encapsulated cells in the air-jet spray method.

  4. H5N1 Vaccine-Elicited Memory B Cells Are Genetically Constrained by the IGHV Locus in the Recognition of a Neutralizing Epitope in the Hemagglutinin Stem.

    Science.gov (United States)

    Wheatley, Adam K; Whittle, James R R; Lingwood, Daniel; Kanekiyo, Masaru; Yassine, Hadi M; Ma, Steven S; Narpala, Sandeep R; Prabhakaran, Madhu S; Matus-Nicodemos, Rodrigo A; Bailer, Robert T; Nabel, Gary J; Graham, Barney S; Ledgerwood, Julie E; Koup, Richard A; McDermott, Adrian B

    2015-07-15

    Because of significant viral diversity, vaccines that elicit durable and broad protection against influenza have been elusive. Recent research has focused on the potential of highly conserved regions of the viral hemagglutinin (HA) as targets for broadly neutralizing Ab responses. Abs that bind the highly conserved stem or stalk of HA can be elicited by vaccination in humans and animal models and neutralize diverse influenza strains. However, the frequency and phenotype of HA stem-specific B cells in vivo remain unclear. In this article, we characterize HA stem-specific B cell responses following H5N1 vaccination and describe the re-expansion of a pre-existing population of memory B cells specific for stem epitopes. This population uses primarily, but not exclusively, IGHV1-69-based Igs for HA recognition. However, within some subjects, allelic polymorphism at the ighv1-69 locus can limit IGHV1-69 immunodominance and may reduce circulating frequencies of stem-reactive B cells in vivo. The accurate definition of allelic selection, recombination requirements, and ontogeny of neutralizing Ab responses to influenza will aid rational influenza vaccine design. Copyright © 2015 by The American Association of Immunologists, Inc.

  5. Reforming of fuel inside fuel cell generator

    Science.gov (United States)

    Grimble, Ralph E.

    1988-01-01

    Disclosed is an improved method of reforming a gaseous reformable fuel within a solid oxide fuel cell generator, wherein the solid oxide fuel cell generator has a plurality of individual fuel cells in a refractory container, the fuel cells generating a partially spent fuel stream and a partially spent oxidant stream. The partially spent fuel stream is divided into two streams, spent fuel stream I and spent fuel stream II. Spent fuel stream I is burned with the partially spent oxidant stream inside the refractory container to produce an exhaust stream. The exhaust stream is divided into two streams, exhaust stream I and exhaust stream II, and exhaust stream I is vented. Exhaust stream II is mixed with spent fuel stream II to form a recycle stream. The recycle stream is mixed with the gaseous reformable fuel within the refractory container to form a fuel stream which is supplied to the fuel cells. Also disclosed is an improved apparatus which permits the reforming of a reformable gaseous fuel within such a solid oxide fuel cell generator. The apparatus comprises a mixing chamber within the refractory container, means for diverting a portion of the partially spent fuel stream to the mixing chamber, means for diverting a portion of exhaust gas to the mixing chamber where it is mixed with the portion of the partially spent fuel stream to form a recycle stream, means for injecting the reformable gaseous fuel into the recycle stream, and means for circulating the recycle stream back to the fuel cells.

  6. Endoderm Generates Endothelial Cells during Liver Development

    Directory of Open Access Journals (Sweden)

    Orit Goldman

    2014-10-01

    Full Text Available Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1. Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.

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

    Directory of Open Access Journals (Sweden)

    Andréa Barbosa de Melo

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

  8. Generation of Mouse Lung Epithelial Cells.

    Science.gov (United States)

    Kasinski, Andrea L; Slack, Frank J

    2013-08-05

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of Kras LSL-G12D/+ ; p53 LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra -G12D and p53 R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  9. Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Abe, Shinji; Takagi, Michiaki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  10. Identification of Potential MHC Class-II-Restricted Epitopes Derived from Leishmania donovani Antigens by Reverse Vaccinology and Evaluation of Their CD4+ T-Cell Responsiveness against Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Manas Ranjan Dikhit

    2017-12-01

    Full Text Available Visceral leishmaniasis (VL is one of the most neglected tropical diseases for which no vaccine exists. In spite of extensive efforts, no successful vaccine is available against this dreadful infectious disease. To support vaccine development, an immunoinformatics approach was applied to screen potential MHC class-II-restricted epitopes that can activate the immune cells. Initially, 37 epitopes derived from six stage-dependent, overexpressed antigens were predicted, which were presented by at least 26 diverse MHC class-II allele. Based on a population coverage analysis and human leukocyte antigen cross-presentation ability, six of the 37 epitopes were selected for further analysis. Stimulation with synthetic peptide alone or as a cocktail triggered intracellular IFN-γ production. Moreover, specific IgG antibodies were detected in the serum of active VL cases against P1, P4, P5, and P6 in order to evaluate the peptide effect on the humoral immune response. Additionally, most of the peptides, except P2, were found to be non-inducers of CD4+ IL-10 against both active VL as well as treated VL subjects. This finding suggests there is no role of these peptides in the pathogenesis of Leishmania. Peptide immunogenicity was validated in BALB/c mice immunized with a cocktail of synthetic peptide emulsified in complete Freund’s adjuvant/incomplete Freund’s adjuvant. The immunized splenocytes induced strong spleen cell proliferation upon parasite re-stimulation. Furthermore, increased IFN-γ, interleukin-12, IL-17, and IL-22 production augmented with elevated nitric oxide (NO synthesis is thought to play a crucial role in macrophage activation. In this investigation, we identified six MHC class-II-restricted epitope hotspots of Leishmania antigens that induce CD4+ Th1 and Th17 responses, which could be used to potentiate a human universal T-epitope vaccine against VL.

  11. Analysis of an immunodominant epitope of topoisomerase I in patients with systemic sclerosis.

    Science.gov (United States)

    Meesters, T M; Hoet, M; van den Hoogen, F H; Verheijen, R; Habets, W J; van Venrooij, W J

    1992-05-01

    In this paper an immunodominant epitope of Topoisomerase I is described. An epitope expression sublibrary was constructed from Topoisomerase I cDNA. The subclones were screened with an antiserum from a patient with systemic sclerosis (SSc). The positive clones defined one immunodominant B cell epitope (epitope III), which was located at the carboxyterminal part of the protein. The epitope, 52 amino acids in length, neither contains the p30gag sequence nor the suggested active site Tyr-723, both presumed antibody recognition sites. More than 70% of our anti-TopoI sera recognize this epitope III, indicating that it is a major recognition site of the anti-TopoI autoantibodies in SSc sera. DNA relaxation experiments show that all sera that recognize epitope III and most sera with antibodies to other epitopes inhibit Topoisomerase I activity.

  12. The Generation and Preclinical Evaulation of Homodimeric Anti-Her-2 Antibodies

    National Research Council Canada - National Science Library

    Vitetta, Ellen

    1999-01-01

    We have generated a panel of 100 anti-Her-2 MAbs and have characterized them with regard to isotype, epitope recognition and ability to signal apoptosis in Her-2 overexpressing prostate cancer cell lines...

  13. The Generation and Preclinical Evaluation of Multimeric Anti-Her-2 Antibodies

    National Research Council Canada - National Science Library

    Vitetta, Ellen

    2000-01-01

    We have generated a panel of 100 anti-Her-2 MAbs and have characterized them with regard to isotype, epitope recognition and ability to signal apoptosis in Her-2-overexpressing breast cancer cell lines...

  14. T cell epitopes of the major fraction of rye grass Lolium perenne (Lol p I) defined using overlapping peptides in vitro and in vivo. I. Isoallergen clone1A.

    Science.gov (United States)

    Bungy Poor Fard, G A; Latchman, Y; Rodda, S; Geysen, M; Roitt, I; Brostoff, J

    1993-10-01

    One hundred and fifteen overlapping synthetic peptides spanning the entire sequence of the iso-allergen clone1A of Lol p I from rye grass Lolium perenne were synthesized by the multi-pin technique. The peptides were overlapping 12mers, offset by two residues and overlapping by 10 residues. Sets of six adjacent overlapping peptides (except pool-1, 15, 20) were pooled and were used in vitro and in vivo to map the T cell epitopes on Lol p I. Six atopics who were skin test and RAST positive to rye grass showed T cell responses to L. perenne extract (LPE) and its major fraction (Lol p I). Five out of six showed T cell responses in vitro to peptide pool-17, while five non-atopics did not respond to any of the peptide pools. By testing the individual peptides of pool-17, we have located the T cell epitope on Lol p I. Interestingly, when we tested pool-17 and its single peptides in vivo by intradermal skin testing we found in one patient a typical DTH after 24-48 h to pool-17 and its peptides (peptides 3 and 4) which exactly matched the in vitro responses. By defining the T cell epitopes in this way a greater understanding of the allergic response to pollen will be obtained, and a more effective and less dangerous vaccine may be possible for treating patients with hay fever.

  15. Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

    Directory of Open Access Journals (Sweden)

    Hatano Manabu

    2004-11-01

    Full Text Available Abstract Background A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. Methods C57BL/6 mice received subcutaneous (s.c. vaccinations with bone marrow-derived dendritic cells (DCs pulsed with synthetic peptides recognized by CD8+ (mEphA2671–679, mEphA2682–689 and CD4+ (mEphA230–44 T cells. Splenocytes (SPCs were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6 establishment/progression was then assessed. Results Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. Conclusions These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells.

  16. SJL mice infected with Acanthamoeba castellanii develop central nervous system autoimmunity through the generation of cross-reactive T cells for myelin antigens.

    Directory of Open Access Journals (Sweden)

    Chandirasegaran Massilamany

    Full Text Available We recently reported that Acanthamoeba castellanii (ACA, an opportunistic pathogen of the central nervous system (CNS possesses mimicry epitopes for proteolipid protein (PLP 139-151 and myelin basic protein 89-101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139-151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that PLP 139-151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139-151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis.

  17. Enhanced immune response and protective effects of nano-chitosan-based DNA vaccine encoding T cell epitopes of Esat-6 and FL against Mycobacterium tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Ganzhu Feng

    Full Text Available Development of a novel and effective vaccine against Mycobacterium tuberculosis (M.tb is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e and fms-like tyrosine kinase 3 ligand (FL genes (termed Esat-6/3e-FL, and was enveloped with chitosan (CS nanoparticles (nano-chitosan. The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against M.tb H37Rv challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing M.tb infection in mice.

  18. Generation of functional organs from stem cells

    Directory of Open Access Journals (Sweden)

    Yunying Liu

    2013-01-01

    Full Text Available We are now well entering the exciting era of stem cells. Potential stem cell therapy holds great promise for the treatment of many diseases such as stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral-sclerosis, myocardial infarction, muscular dystrophy, diabetes, and etc. It is generally believed that transplantation of specific stem cells into the injured tissue to replace the lost cells is an effective way to repair the tissue. In fact, organ transplantation has been successfully practiced in clinics for liver or kidney failure. However, the severe shortage of donor organs has been a major obstacle for the expansion of organ transplantation programs. Toward that direction, generation of transplantable organs using stem cells is a desirable approach for organ replacement and would be of great interest for both basic and clinical scientists. Here we review recent progress in the field of organ generation using various methods including single adult tissue stem cells, a blastocyst complementation system, tissue decellularization/recellularization and a combination of stem cells and tissue engineering.

  19. Peptides derived from self-proteins as partial agonists and antagonists of human CD8+ T-cell clones reactive to melanoma/melanocyte epitope MART1(27-35)

    DEFF Research Database (Denmark)

    Loftus, D J; Squarcina, P; Nielsen, M B

    1998-01-01

    The self-peptide MART1(27-35) derives from the melanocyte/melanoma protein Melan A/MART1 and is a target epitope of CD8+ T cells, commonly recovered from tumor-infiltrating lymphocytes of HLA-A2.1+ melanoma patients. Despite their prevalence in such patients, these CTLs generally appear to be ine......The self-peptide MART1(27-35) derives from the melanocyte/melanoma protein Melan A/MART1 and is a target epitope of CD8+ T cells, commonly recovered from tumor-infiltrating lymphocytes of HLA-A2.1+ melanoma patients. Despite their prevalence in such patients, these CTLs generally appear...

  20. Establishment of HLA-DR4 transgenic mice for the identification of CD4+ T cell epitopes of tumor-associated antigens.

    Directory of Open Access Journals (Sweden)

    Junji Yatsuda

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

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

    Science.gov (United States)

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

    2017-02-01

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

  2. A novel recombinant peptide containing only two T-cell tolerance epitopes of chicken type II collagen that suppresses collagen-induced arthritis.

    Science.gov (United States)

    Xi, Caixia; Tan, Liuxin; Sun, Yeping; Liang, Fei; Liu, Nan; Xue, Hong; Luo, Yuan; Yuan, Fang; Sun, Yuying; Xi, Yongzhi

    2009-02-01

    Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50 microg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50 microg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-gamma level, and increase Th3-cytokine TGF-beta(1) produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2(b) MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA.

  3. Generation of T cell help through a MHC class I-restricted TCR

    NARCIS (Netherlands)

    Kessels, Helmut W. H. G.; Schepers, Koen; van den Boom, Marly D.; Topham, David J.; Schumacher, Ton N. M.

    2006-01-01

    CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine

  4. Loss of immunization-induced epitope-specific CD4 T-cell response following anaplasma marginale infection requires presence of the T-cell epitope on the pathogen and is not associated with an increase in lymphocytes

    Science.gov (United States)

    We have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginale induces a functionally exhausted CD4 T-cell response to the A. marginale immunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were...

  5. TCLP: an online cancer cell line catalogue integrating HLA type, predicted neo-epitopes, virus and gene expression.

    Science.gov (United States)

    Scholtalbers, Jelle; Boegel, Sebastian; Bukur, Thomas; Byl, Marius; Goerges, Sebastian; Sorn, Patrick; Loewer, Martin; Sahin, Ugur; Castle, John C

    2015-11-20

    Human cancer cell lines are an important resource for research and drug development. However, the available annotations of cell lines are sparse, incomplete, and distributed in multiple repositories. Re-analyzing publicly available raw RNA-Seq data, we determined the human leukocyte antigen (HLA) type and abundance, identified expressed viruses and calculated gene expression of 1,082 cancer cell lines. Using the determined HLA types, public databases of cell line mutations, and existing HLA binding prediction algorithms, we predicted antigenic mutations in each cell line. We integrated the results into a comprehensive knowledgebase. Using the Django web framework, we provide an interactive user interface with advanced search capabilities to find and explore cell lines and an application programming interface to extract cell line information. The portal is available at http://celllines.tron-mainz.de.

  6. Simultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti- CD3 mAb T-cell expansion and "RecycleSpot"

    Directory of Open Access Journals (Sweden)

    Wong Johnson T

    2005-05-01

    Full Text Available Abstract The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot, further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  8. Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads

    Science.gov (United States)

    Salvat, Regina S.; Choi, Yoonjoo; Bishop, Alexandra; Bailey-Kellogg, Chris; Griswold, Karl E.

    2015-01-01

    Anti-drug immune responses are a unique risk factor for biotherapeutics, and undesired immunogenicity can alter pharmacokinetics, compromise drug efficacy, and in some cases even threaten patient safety. To fully capitalize on the promise of biotherapeutics, more efficient and generally applicable protein deimmunization tools are needed. Mutagenic deletion of a protein’s T cell epitopes is one powerful strategy to engineer immunotolerance, but deimmunizing mutations must maintain protein structure and function. Here, EpiSweep, a structure-based protein design and deimmunization algorithm, has been used to produce a panel of seven beta-lactamase drug candidates having 27–47% reductions in predicted epitope content. Despite bearing eight mutations each, all seven engineered enzymes maintained good stability and activity. At the same time, the variants exhibited dramatically reduced interaction with human class II major histocompatibility complex proteins, key regulators of anti-drug immune responses. When compared to 8-mutation designs generated with a sequence-based deimmunization algorithm, the structure-based designs retained greater thermostability and possessed fewer high affinity epitopes, the dominant drivers of anti-biotherapeutic immune responses. These experimental results validate the first structure-based deimmunization algorithm capable of mapping optimal biotherapeutic design space. By designing optimal mutations that reduce immunogenic potential while imparting favorable intramolecular interactions, broadly distributed epitopes may be simultaneously targeted using high mutational loads. PMID:25655032

  9. Internal and ancestral controls of cell-generation times

    Science.gov (United States)

    Kubitschek, H. E.

    1969-01-01

    Lateral and longitudinal correlations between related cells reveal associations between the generation times of cells for an intermediate period /three generations in bacteral cultures/. Generation times of progeny are influenced by nongenetic factors transmitted from their ancestors.

  10. Fuel cells for distributed power generation

    Science.gov (United States)

    Tarman, Paul B.

    Deregulation has caused a major change in power distribution in the USA. Large central power stations are being and will continue to be replaced by smaller, distributed power generation sources of less than 20 kW. Fuel cells, specifically molten carbonate fuel cells (MCFCs), are best suited to serve this need. Small turbines cannot achieve the efficiency or environmental friendliness of MCFCs in this power range. This paper discusses the goals of M-C Power Corporation and the advantages of its IMHEX® MCFC technology. M-C Power's factory, demonstration testing program, and its market-entry power plant are also described, as are its commercialization strategy and schedule.

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

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    2010-07-01

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

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

    Science.gov (United States)

    Hunt, Ann R; Frederickson, Shana; Maruyama, Toshiaki; Roehrig, John T; Blair, Carol D

    2010-07-13

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

  13. Recombinant influenza viruses as delivery vectors for hepatis B virus epitopes.

    Science.gov (United States)

    Song, Jae-Min; Lee, Kwang-Hee; Seong, Baik-Lin

    2012-07-01

    Neuraminidase (NA) of influenza virus contains stalk region that shows a great deal of variability in both amino acid sequence and length. In this paper, we investigated generation of recombinant influenza viruses that had hepatitis B virus (HBV) B cell epitopes in the NA stalk region as a dual vaccine candidate. We used the WSH-HK reassortant helper virus for rescue of recombinant influenza virus containing HBV epitopes and reverse genetic protocol based on the use of micrococcal nuclease-treated virus cores for reconstitution of ribonucleoproteins. We successfully generated a chimeric influenza viruses which contained 22 amino acid peptides in the stalk region derived from the surface and pre-surface protein HBV. The growth kinetics of the recombinant viruses was investigated after infection of Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney (MDBK) cells and the rIV-BVPreS virus showed higher titer than other viruses in MDCK cells. We also confirmed the presence of HBV epitopes in the chimeric viruses by enzyme-linked immunosorbent assay (ELISA) using anti-HBV polyclonal antibody. When the ratio of recombinant virus verse wild type virus was calculated by ELISA, recombinant viruses exhibited 2 fold higher values than the wild type virus. These results suggest that chimeric influenza virus which contained foreign antigens can be used as dual vaccine against both HBV and influenza viruses.

  14. The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies▿

    Science.gov (United States)

    Zhang, Jun; Rao, Eileen; Dioszegi, Marianna; Kondru, Rama; DeRosier, Andre; Chan, Eva; Schwoerer, Stephan; Cammack, Nick; Brandt, Michael; Sankuratri, Surya; Ji, Changhua

    2007-01-01

    Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2. PMID:17242138

  15. Navigating diabetes-related immune epitope data: resources and tools provided by the Immune Epitope Database (IEDB)

    Science.gov (United States)

    Vaughan, Kerrie; Peters, Bjoern; Mallone, Roberto; von Herrath, Matthias; Roep, Bart O.; Sette, Alessandro

    2014-01-01

    Background The Immune Epitope Database (IEDB), originally focused on infectious diseases, was recently expanded to allergy, transplantation and autoimmunity diseases. Here we focus on diabetes, chosen as a prototype autoimmune disease. We utilize a combined tutorial and meta-analysis format, which demonstrates how common questions, related to diabetes epitopes can be answered. Results A total of 409 references are captured in the IEDB describing >2,500 epitopes from diabetes associated antigens. The vast majority of data were derived from GAD, insulin, IA-2/PTPRN, IGRP, ZnT8, HSP, and ICA-1, and the experiments related to T cell epitopes and MHC binding far outnumbers B cell assays. We illustrate how to search by specific antigens, epitopes or host. Other examples include searching for tetramers or epitopes restricted by specific alleles or assays of interest, or searching based on the clinical status of the host. Conclusions The inventory of all published diabetes epitope data facilitates its access for the scientific community. While the global collection of primary data from the literature reflects potential investigational biases present in the literature, the flexible search approach allows users to perform queries tailored to their preferences, including or excluding data as appropriate. Moreover, the analysis highlights knowledge gaps and identifies areas for future investigation. PMID:25140192

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-28

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

  17. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

    Science.gov (United States)

    Zheng, Long; Hu, Peisheng; Wolfe, Brandon; Gonsalves, Caryn; Ren, Luqing; Khawli, Leslie A; Kaslow, Harvey R; Epstein, Alan L

    2017-12-20

    T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgamma null (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

  18. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma

    Directory of Open Access Journals (Sweden)

    Long Zheng

    2017-12-01

    Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

  19. Binding of human beta 2-microglobulin to murine EL4 thymoma cells upregulates MHC class I heavy-chain epitopes, inhibits IL-2 secretion and induces resistance to killing by natural killer cells

    DEFF Research Database (Denmark)

    Claësson, M H; Nissen, Mogens Holst

    1994-01-01

    A variety of murine tumor cell lines was studied for its binding of exogeneously added human beta 2-microglobulin (h beta 2m). Three T lymphomas and one IL-2-dependent T-cell line (HT-1) bound substantial amounts of h beta 2m, whereas P815 mastocytoma cells, an Abelson virus-infected pre-B cell...... line (ABLS-8), X63 B-lymphoma cells and YAC cells did not bind h beta 2m. In two of the T lymphomas, EL4 and BW5147, binding of h beta 2m led to an increase in major histocompatibility complex class I (MHC-I) heavy-chain epitope expression as measured by anti-H-2K/D antibody binding and FACS analysis....... EL4 cells which had bound h beta 2m decreased their rate of constitutive IL-2 secretion and became resistant to activated natural killer (NK) cell killing. The present data suggest the binding of h beta 2m to mouse T cells leads to conformational changes of MHC-I heavy chains which influence both...

  20. Immunogenicity of novel Dengue virus epitopes identified by bioinformatic analysis.

    Science.gov (United States)

    Sánchez-Burgos, Gilma; Ramos-Castañeda, José; Cedillo-Rivera, Roberto; Dumonteil, Eric

    2010-10-01

    We used T cell epitope prediction tools to identify epitopes from Dengue virus polyprotein sequences, and evaluated in vivo and in vitro the immunogenicity and antigenicity of the corresponding synthetic vaccine candidates. Twenty-two epitopes were predicted to have a high affinity for MHC class I (H-2Kd, H-2Dd, H-2Ld alleles) or class II (IAd alleles). These epitopes were conserved between the four virus serotypes, but with no similarity to human and mouse sequences. Thirteen synthetic peptides induced specific antibodies production with or without T cells activation in mice. Three synthetic peptides induced mostly IgG antibodies, and one of these from the E gene induced a neutralizing response. Ten peptides induced a combination of humoral and cellular responses by CD4+ and CD8+ T cells. Twelve peptides were novel B and T cell epitopes. These results indicate that our bioinformatics strategy is a powerful tool for the identification of novel antigens and its application to human HLA may lead to a potent epitope-based vaccine against Dengue virus and many other pathogens. (c) 2010 Elsevier B.V. All rights reserved.

  1. Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection

    Science.gov (United States)

    Zhang, Wei; Lonning, Scott M.; McGuire, Travis C.

    1998-01-01

    Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV. PMID:9811694

  2. Gag protein epitopes recognized by ELA-A-restricted cytotoxic T lymphocytes from horses with long-term equine infectious anemia virus infection.

    Science.gov (United States)

    Zhang, W; Lonning, S M; McGuire, T C

    1998-12-01

    Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.

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

    Directory of Open Access Journals (Sweden)

    Widodo

    2018-03-01

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

  4. Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes

    DEFF Research Database (Denmark)

    Fomsgaard, A; Nielsen, H V; Kirkby, N

    1999-01-01

    Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high...... degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible......'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu...

  5. Rapid progressing allele HLA-B35 Px restricted anti-HIV-1 CD8+ T cells recognize vestigial CTL epitopes.

    Directory of Open Access Journals (Sweden)

    Christian B Willberg

    Full Text Available The HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele.Immune responses to two HLA-B*35 restricted HIV-1 specific CTL epitopes and their variants were followed longitudinally during early HIV-1 infection in 16 HLA-B*35+ individuals. Subjects expressing HLA-B*35-Px alleles showed no difference in response to the consensus epitopes compared to individuals with HLA-B*35-Py alleles. Surprisingly, all the HLA-B*35-Px+ individuals responded to epitope-variants even in the absence of a consensus response. Sequencing of the viral population revealed no evidence of variant virus in any of the individuals.This demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.

  6. Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA

    DEFF Research Database (Denmark)

    Barfod, L.; Bernasconi, N. L.; Dahlback, M.

    2007-01-01

    -molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-epsilon domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates...

  7. 76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

    Science.gov (United States)

    2011-08-18

    ..., MD 20892, Telephone: 301-827-0793; E-mail: [email protected] . For Financial and... requires extensive infrastructure for growing cells, purifying HLA from culture supernatants, and for mass... allow FDA to acquire the proteomic expertise, training, and tissue culture support to establish a...

  8. Heparin increases the infectivity of Human Papillomavirus type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

    NARCIS (Netherlands)

    Cerqueira, C.; Liu, Y.; Kuhling, L.; Chai, W.; Hafezi, W.; Kuppevelt, T.H. van; Kuhn, J.E.; Feizi, T.; Schelhaas, M.

    2013-01-01

    Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin

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

    Science.gov (United States)

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

    2018-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Caixia Gao

    2017-12-01

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

  11. Mapping of T cell epitopes of the major fraction of rye grass using peripheral blood mononuclear cells from atopics and non-atopics. II. Isoallergen clone 5A of Lolium perenne group I (Lol p I).

    Science.gov (United States)

    Bungy, G A; Rodda, S; Roitt, I; Brostoff, J

    1994-09-01

    Rye grass is the major cause of hay fever which currently affects 20% of the population. Lolium perenne group I (Lol p I) is a glycoprotein of 240 amino acid residues, representing the main allergen of rye grass. We have used peripheral blood mononuclear cells (PBMC) from controls and subjects allergic to rye grass and cultured them with L. perenne extract (LPE) and Lol p I and measured lymphocyte activation using thymidine incorporation. Patients were further studied against the 115 overlapping peptides of the iso-allergen clone 5A of Lol p I to see whether the 4 amino acid residue differences between clone 1A and clone 5A affect the T cell epitope and thus, lymphocyte activation. There are 24 peptide differences between isoallergen clone 1A and clone 5A occurring in pools 4, 13, 16 and 19 each one of which could be an immunodominant epitope. The PBMC from all allergic patients studied showed a strong proliferative response to LPE and Lol p I. Five immunogenic peptide pools, pool 6, 15, 16, 17 and 19 of the isoallergen clone 5A were also identified. Most of these pools are in the C-terminal region of Lol p I. Out of 20 pools tested in vitro 1 pool (pool-17) induced PBMC proliferation in five out of six patients who were not restricted to an HLA class II DR gene product. However, three out of the six subjects responded to various other peptide pools in addition to the immunodominant pool. In spite of the amino acid differences between the two clones, pool 17 still remains the immunodominant T cell epitope. Control subjects showed only weak responses to LPE and no detectable response to either Lol p I or peptide pools. From within the most active pool we have defined two peptides of the isoallergen clone 5A (identical in sequence with clone 1A) which stimulate lymphocytes from rye grass-sensitive patients in vitro. Previous studies with the two continuous sequences (193WGAVWRIDTPDK204 and 195AVWRIDTPDKLT206) tested in vivo by intradermal skin testing have shown

  12. Epitope prediction methods

    DEFF Research Database (Denmark)

    Karosiene, Edita

    on machine learning techniques. Several MHC class I binding prediction algorithms have been developed and due to their high accuracy they are used by many immunologists to facilitate the conventional experimental process of epitope discovery. However, the accuracy of these methods depends on data defining...... the NetMHCIIpan-3.0 predictor based on artificial neural networks, which is capable of giving binding affinities to any human MHC class II molecule. Chapter 4 of this thesis gives an overview of bioinformatics tools developed by the Immunological Bioinformatics group at Center for Biological Sequence...

  13. Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8+ T-Cell Epitope in p1 Gag Protein▿

    Science.gov (United States)

    Prado, Julia G.; Honeyborne, Isobella; Brierley, Ian; Puertas, Maria Carmen; Martinez-Picado, Javier; Goulder, Philip J. R.

    2009-01-01

    The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI429-437 (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness. PMID:18945768

  14. Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein.

    Science.gov (United States)

    Prado, Julia G; Honeyborne, Isobella; Brierley, Ian; Puertas, Maria Carmen; Martinez-Picado, Javier; Goulder, Philip J R

    2009-01-01

    The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI(429-437) (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.

  15. Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Ngoc-Luong; So, Kum-Kang; Kim, Jung-Mi; Kim, Sae-Hae; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

    2015-01-01

    A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S. cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1-0.2% of cell-free extracts (CFEs). In addition, 0.1-0.2 mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Pronase E-Based Generation of Fluorescent Peptide Fragments: Tracking Intracellular Peptide Fate in Single Cells.

    Science.gov (United States)

    Mainz, Emilie R; Dobes, Nicholas C; Allbritton, Nancy L

    2015-08-04

    The ability to track intracellular peptide proteolysis at the single cell level is of growing interest, particularly as short peptide sequences continue to play important roles as biosensors, therapeutics, and endogenous participants in antigen processing and intracellular signaling. We describe a rapid and inexpensive methodology to generate fluorescent peptide fragments from a parent sequence with diverse chemical properties, including aliphatic, nonpolar, basic, acidic, and non-native amino acids. Four peptide sequences with existing biochemical applications were fragmented using incubation with Pronase E and/or formic acid, and in each case a complete set of fluorescent fragments was generated for use as proteolysis standards in chemical cytometry. Fragment formation and identity was monitored with capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-MS) to confirm the presence of all sequences and yield fragmentation profiles across Pronase E concentrations which can readily be used by others. As a pilot study, Pronase E-generated standards from an Abl kinase sensor and an ovalbumin antigenic peptide were then employed to identify proteolysis products arising from the metabolism of these sequences in single cells. The Abl kinase sensor fragmented at 4.2 ± 4.8 zmol μM(-1) s(-1) and the majority of cells possessed similar fragment identities. In contrast, an ovalbumin epitope peptide was degraded at 8.9 ± 0.1 zmol μM(-1) s(-1), but with differential fragment formation between individual cells. Overall, Pronase E-generated peptide standards were a rapid and efficient method to identify proteolysis products from cells.

  17. Immunoglobulins against Tyrosine Nitrated Epitopes in Coronary Artery Disease

    Science.gov (United States)

    Thomson, Leonor; Tenopoulou, Margarita; Lightfoot, Richard; Tsika, Epida; Parastatidis, Ioannis; Martinez, Marissa; Greco, Todd M.; Doulias, Paschalis-Thomas; Wu, Yuping; Tang, W. H. Wilson; Hazen, Stanley L.; Ischiropoulos, Harry

    2012-01-01

    Background Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine nitrated proteins, a footprint of oxygen and nitrogen derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of coronary artery disease (CAD) subjects. However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of CAD subjects. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in CAD patients (n=374) and subjects without CAD (non CAD controls, n=313). A ten-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in the CAD subjects (3.75 ± 1.8 μg antibody Eq/mL plasma vs. 0.36 ± 0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions The results of this cross sectional study suggest that post-translational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neoepitopes for elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD. PMID:23081989

  18. Generation of hematopoietic lineage cells from embryonic like cells

    Directory of Open Access Journals (Sweden)

    Gholam Reza Khamisipour

    2014-10-01

    Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

  19. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  20. Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules

    DEFF Research Database (Denmark)

    Joosten, I; Wauben, M H; Holewijn, M C

    1994-01-01

    characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides......New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one...... must be able to assess peptide-MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptides. So far no information has been available on the peptide binding...

  1. Experimental validation of multi-epitope peptides including promising MHC class I- and class II-restricted epitopes of four known Leishmania infantum proteins

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    Maria eAgallou

    2014-06-01

    Full Text Available Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4+ and CD8+ T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania (L. infantum proteins, CPA, histone H1, KMP-11 and LeIF were analysed for the prediction of binding epitopes to H2d MHC class I and class II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and class II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in CFA/IFA. CPA_p2, CPA_p3, H1_p1 and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3 and LeIF_p6 induced IFN-γ-producing CD4+ T cells indicating a TH1 type response. In addition, CPA_p2, CPA_p3 and H1_p1 induced also the induction of CD8+ T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3 and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

  2. An Arthritis-Suppressive and Treg Cell-Inducing CD4+ T Cell Epitope is Functional in the Context of HLA-Restricted T Cell Responses

    NARCIS (Netherlands)

    De Wolf, Charlotte; Van Der Zee, Ruurd; Den Braber, Ineke; Glant, Tibor; Maill??re, Bernard; Favry, Emmanuel; Van Lummel, Menno; Koning, Frits; Hoek, Aad; Ludwig, Irene; Van Eden, Willem; Broere, Femke

    2016-01-01

    Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding

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

    Directory of Open Access Journals (Sweden)

    Encheng Sun

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

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

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    Denicar Lina Nascimento Fabris Maeda

    2017-09-01

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

  5. A Fast and Inexpensive Protocol for Empirical Verification of Neutralizing Epitopes in Microbial Toxins and Enzymes

    Directory of Open Access Journals (Sweden)

    Christine N. Vuong

    2017-06-01

    Full Text Available In vivo targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, Clostridium perfringens alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected in silico using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for in vitro toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin’s hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and de facto identification of neutralizing epitopes in microbial

  6. Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

    Science.gov (United States)

    Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Hoshino, Yasutaka; Yuan, Lijuan

    2015-01-01

    The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

  7. Computer aided epitope design as a peptide vaccine component against Lassa virus.

    Science.gov (United States)

    Faisal, Ar-Rafi Md; Imtiaz, Syed Hassan; Zerin, Tasnim; Rahman, Tania; Shekhar, Hossain Uddin

    2017-01-01

    Lassa virus (LASV) is an arena virus causing hemorrhagic fever and it is endemic in several regions of West Africa. The disease-causing virus records high mortality rate in endemic regions due to lack of appropriate treatment and prevention strategies. Therefore, it is of interest to design and develop viable vaccine components against the virus. We used the Lassa virus envelope glyco-proteins as a vaccine target to identify linear peptides as potential epitopes with immunogenic properties by computer aided epitope prediction tools. We report a T-cell epitope 'LLGTFTWTL' and a B-cell epitope 'AELKCFGNTAVAKCNE' with predicted potential immunogenicity for further in vivo and in vitro consideration.

  8. Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

    Science.gov (United States)

    Wen, Xiaobo; Wen, Ke; Cao, Dianjun; Li, Guohua; Jones, Ronald W; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka; Yuan, Lijuan

    2014-07-31

    Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 μg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 μg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or

  9. Treatment with chemotherapy and dendritic cells pulsed with multiple Wilms' tumor 1 (WT1)-specific MHC class I/II-restricted epitopes for pancreatic cancer.

    Science.gov (United States)

    Koido, Shigeo; Homma, Sadamu; Okamoto, Masato; Takakura, Kazuki; Mori, Masako; Yoshizaki, Shinji; Tsukinaga, Shintaro; Odahara, Shunichi; Koyama, Seita; Imazu, Hiroo; Uchiyama, Kan; Kajihara, Mikio; Arakawa, Hiroshi; Misawa, Takeyuki; Toyama, Yoichi; Yanagisawa, Satoru; Ikegami, Masahiro; Kan, Shin; Hayashi, Kazumi; Komita, Hideo; Kamata, Yuko; Ito, Masaki; Ishidao, Takefumi; Yusa, Sei-Ichi; Shimodaira, Shigetaka; Gong, Jianlin; Sugiyama, Haruo; Ohkusa, Toshifumi; Tajiri, Hisao

    2014-08-15

    We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy. Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II-restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine. The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4(+) T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days. The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer. ©2014 American Association for Cancer Research.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4...... by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes....

  11. De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes.

    Science.gov (United States)

    Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

    2015-01-01

    Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.

  12. Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the timothy grass major allergen Phl p 5a

    DEFF Research Database (Denmark)

    Hecker, J.; Diethers, A.; Seismann, H.

    2011-01-01

    and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain...

  13. High Throughput T Epitope Mapping and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Giuseppina Li Pira

    2010-01-01

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

  14. EpitopeViewer: a Java application for the visualization and analysis of immune epitopes in the Immune Epitope Database and Analysis Resource (IEDB)

    Science.gov (United States)

    Beaver, John E; Bourne, Philip E; Ponomarenko, Julia V

    2007-01-01

    Background Structural information about epitopes, particularly the three-dimensional (3D) structures of antigens in complex with immune receptors, presents a valuable source of data for immunology. This information is available in the Protein Data Bank (PDB) and provided in curated form by the Immune Epitope Database and Analysis Resource (IEDB). With continued growth in these data and the importance in understanding molecular level interactions of immunological interest there is a need for new specialized molecular visualization and analysis tools. Results The EpitopeViewer is a platform-independent Java application for the visualization of the three-dimensional structure and sequence of epitopes and analyses of their interactions with antigen-specific receptors of the immune system (antibodies, T cell receptors and MHC molecules). The viewer renders both 3D views and two-dimensional plots of intermolecular interactions between the antigen and receptor(s) by reading curated data from the IEDB and/or calculated on-the-fly from atom coordinates from the PDB. The 3D views and associated interactions can be saved for future use and publication. The EpitopeViewer can be accessed from the IEDB Web site through the quick link 'Browse Records by 3D Structure.' Conclusion The EpitopeViewer is designed and been tested for use by immunologists with little or no training in molecular graphics. The EpitopeViewer can be launched from most popular Web browsers without user intervention. A Java Runtime Environment (RJE) 1.4.2 or higher is required. PMID:17313688

  15. CO-GENERATION AND OPERATING NETWORK CELLS

    DEFF Research Database (Denmark)

    Nielsen, John Eli

    2008-01-01

    In Denmark several thousands of generators are connected to the distribution system (10 kV and 0.4 kV). The production from these generators many times exceeds the load. The generators can be divided into two types, Wind turbines and CHP generators. These generators have one thing in common......, the power system they are connected to, has never been designed to accommodate so many generators. In Denmark we now expect a third type of generators: the microgenerators. This time we want to be prepared. Denmark therefore now participates in a lot of research and full scale demonstration projects. A key...

  16. [Construction and characterization of an epitope-mutated Asia 1 type foot-and-mouth disease virus].

    Science.gov (United States)

    Zhang, Yan; Hu, Yonghao; Yang, Fan; Yang, Bo; Wang, Songhao; Zhu, Zixiang; Zheng, Haixue

    2015-01-01

    To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes...... in subcutaneous tumors revealed that survivin-specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two K(b)-restricted peptide epitopes derived from the murine survivin protein; moreover, survivin-specific vaccination not only resulted...

  18. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

    Directory of Open Access Journals (Sweden)

    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  19. Human immune responses to H. pylori HLA Class II epitopes identified by immunoinformatic methods.

    Directory of Open Access Journals (Sweden)

    Songhua Zhang

    Full Text Available H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.

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

    Science.gov (United States)

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

    2018-04-02

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

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

    Science.gov (United States)

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

    2017-12-01

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

  2. AC power generation from microbial fuel cells

    Science.gov (United States)

    Lobo, Fernanda Leite; Wang, Heming; Forrestal, Casey; Ren, Zhiyong Jason

    2015-11-01

    Microbial fuel cells (MFCs) directly convert biodegradable substrates to electricity and carry good potential for energy-positive wastewater treatment. However, the low and direct current (DC) output from MFC is not usable for general electronics except small sensors, yet commercial DC-AC converters or inverters used in solar systems cannot be directly applied to MFCs. This study presents a new DC-AC converter system for MFCs that can generate alternating voltage in any desired frequency. Results show that AC power can be easily achieved in three different frequencies tested (1, 10, 60 Hz), and no energy storage layer such as capacitors was needed. The DC-AC converter efficiency was higher than 95% when powered by either individual MFCs or simple MFC stacks. Total harmonic distortion (THD) was used to investigate the quality of the energy, and it showed that the energy could be directly usable for linear electronic loads. This study shows that through electrical conversion MFCs can be potentially used in household electronics for decentralized off-grid communities.

  3. New generation of dendritic cell vaccines.

    Science.gov (United States)

    Radford, Kristen J; Caminschi, Irina

    2013-02-01

    Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses, including the induction of cytotoxic T lymphocytes (CTL) responses. These are essential for the eradication of cancers and pathogens including HIV and malaria, for which there are currently no effective vaccines. New developments in our understanding of DC biology have identified the key DC subset responsible for CTL induction, which is now an attractive candidate to target for vaccination. These DC are characterized by expression of novel markers Clec9A and XCR1, and a specialized capacity to cross-present antigen (Ag) from tumors and pathogens that do not directly infect DC. New generation DC vaccines that specifically target the cross-presenting DC in vivo have already demonstrated potential in preclinical animal models but the challenge remains to translate these findings into clinically efficacous vaccines in man. This has been greatly facilitated by the recent identification of the equivalent Clec9A(+) XCR1(+) cross-presenting DC in human lymphoid tissues and peripheral tissues that are key sites for vaccination administration. These findings combined with further studies on DC subset biology have important implications for the design of new CTL-mediated vaccines.

  4. Antibody Production and Th1-biased Response Induced by an Epitope Vaccine Composed of Cholera Toxin B Unit and Helicobacter pylori Lpp20 Epitopes.

    Science.gov (United States)

    Li, Yan; Chen, Zhongbiao; Ye, Jianbin; Ning, Lijun; Luo, Jun; Zhang, Lili; Jiang, Yin; Xi, Yue; Ning, Yunshan

    2016-06-01

    The epitope vaccine is an attractive potential for prophylactic and therapeutic vaccination against Helicobacter pylori (H. pylori) infection. Lpp20 is one of major protective antigens which trigger immune response after H. pylori invades host and has been considered as an excellent vaccine candidate for the control of H. pylori infection. In our previous study, one B-cell epitope and two CD4(+) T-cell epitopes of Lpp20 were identified. In this study, an epitope vaccine composed of mucosal adjuvant cholera toxin B subunit (CTB) and these three identified Lpp20 epitopes were constructed to investigate the efficacy of this epitope vaccine in mice. The epitope vaccine including CTB, one B-cell, and two CD4(+) T-cell epitopes of Lpp20 was constructed and named CTB-Lpp20, which was then expressed in Escherichia coli and used for intraperitoneal immunization in BALB/c mice. The immunogenicity, specificity, and ability to induce antibodies against Lpp20 and cytokine secretion were evaluated. After that, CTB-Lpp20 was intragastrically immunized to investigate the prophylactic and therapeutic efficacy in infected mice. The results indicated that the epitope vaccine CTB-Lpp20 possessed good immunogenicity and immunoreactivity and could elicit specific high level of antibodies against Lpp20 and the cytokine of IFN-γ and IL-17. Additionally, CTB-Lpp20 significantly decreased H. pylori colonization in H. pylori challenging mice, and the protection was correlated with IgG, IgA, and sIgA antibody and Th1-type cytokines. This study will be better for understanding the protective immunity of epitope vaccine, and CTB-Lpp20 may be an alternative strategy for combating H. pylori invasion. © 2015 John Wiley & Sons Ltd.

  5. Frequency of Interferon-Resistance Conferring Substitutions in Amino Acid Positions 70 and 91 of Core Protein of the Russian HCV 1b Isolates Analyzed in the T-Cell Epitopic Context

    Directory of Open Access Journals (Sweden)

    V. S. Kichatova

    2018-01-01

    Full Text Available Amino acid substitutions R70Q/H and L91M in HCV subtype 1b core protein can affect the response to interferon and are associated with the development of hepatocellular carcinoma. We found that the rate of R70Q/H in HCV 1b from Russia was 31.2%, similar to that in HCV strains from Asia (34.0%, higher than that in the European (18.0%, p=0.0010, but lower than that in the US HCV 1b strains (62.8%, p<0.0001. Substitution L91M was found in 80.4% of the Russian HCV 1b isolates, higher than in Asian isolates (43.8%, p<0.0001. Thus, a significant proportion of Russian HCV 1b isolates carry the unfavorable R70Q/H and/or L91M substitution. In silico analysis of the epitopic structure of the regions of substitutions revealed that both harbor clusters of T-cell epitopes. Peptides encompassing these regions were predicted to bind to a panel of HLA class I molecules, with substitutions impairing peptide recognition by HLA I molecules of the alleles prevalent in Russia. This indicates that HCV 1b with R70Q/H and L91M substitutions may have evolved as the immune escape variants. Impairment of T-cell recognition may play a part in the negative effect of these substitutions on the response to IFN treatment.

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

    Directory of Open Access Journals (Sweden)

    Nathali Kaushansky

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

  7. Organization of haemopoietic stem cells: the generation-age hypothesis

    International Nuclear Information System (INIS)

    Rosendaal, M.; Hodgson, G.S.; Bradley, T.R.

    1978-01-01

    This paper proposes that the previous division history of each stem cell is one determinant of the functional organisation of the haemopoietic stem cell population. Older stem cell are used to form blood before younger ones. The stem cells generating capacity of a lineage is finite, and cells are eventually lost to the system by forming two committed precursors of the cell lines, and the next oldest stem cell takes over. Hence the proposed term 'generation-age hypothesis', supported by experimental evidence. Older stem cells from normal bone marrow and 13 day foetal liver were stripped away with phase-specific drugs revealing a younger population of stem cells with three-to four-fold greater stem cell generating capacity. Normal stem cells aged by continuous irradiation and serial retransplantation had eight-fold reduced generating capacity. That of stem cells in the bloodstream was half to a quarter that of normal bone marrow stem cells. There were some circulating stem cells, identified by reaction to brain-associated antigen, positive for 75% of normal femoral stem cells but not their progeny, whose capacity for stem cell generation was an eighth to one fortieth that of normal cells. (U.K.)