WorldWideScience

Sample records for acid decarboxylase-derived epitopes

  1. Branched-chain 2-keto acid decarboxylases derived from Psychrobacter.

    Science.gov (United States)

    Wei, Jiashi; Timler, Jacobe G; Knutson, Carolann M; Barney, Brett M

    2013-09-01

    The conversion of branched-chain amino acids to branched-chain acids or alcohols is an important aspect of flavor in the food industry and is dependent on the Ehrlich pathway found in certain lactic acid bacteria. A key enzyme in the pathway, the 2-keto acid decarboxylase (KDC), is also of interest in biotechnology applications to produce small branched-chain alcohols that might serve as improved biofuels or other commodity feedstocks. This enzyme has been extensively studied in the model bacterium Lactococcus lactis, but is also found in other bacteria and higher organisms. In this report, distinct homologs of the L. lactis KDC originally annotated as pyruvate decarboxylases from Psychrobacter cryohalolentis K5 and P. arcticus 273-4 were cloned and characterized, confirming a related activity toward specific branched-chain 2-keto acids derived from branched-chain amino acids. Further, KDC activity was confirmed in intact cells and cell-free extracts of P. cryohalolentis K5 grown on both rich and defined media, indicating that the Ehrlich pathway may also be utilized in some psychrotrophs and psychrophiles. A comparison of the similarities and differences in the P. cryohalolentis K5 and P. arcticus 273-4 KDC activities to other bacterial KDCs is presented. PMID:23826991

  2. Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

    OpenAIRE

    Xu, W; Ellington, A. D.

    1996-01-01

    In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of huma...

  3. Lipophosphoglycan and secreted acid phosphatase of Leishmania tropica share species-specific epitopes.

    Science.gov (United States)

    Jaffe, C L; Perez, L; Schnur, L F

    1990-06-01

    Several species-specific monoclonal antibodies (T11, T13-T15) which only react with Leishmania tropica, recognize phosphorlated carbohydrate epitopes on lipophosphoglycan and the structurally related molecule, phosphoglycan, which is shed by promastigotes into spent culture medium. During immunoaffinity isolation of [32P]orthophosphate-labeled phosphoglycan on monoclonal antibody T15 conjugated to Sepharose 4B, a high-Mr component (approx. 200,000) was co-purified. The latter material is metabolically labeled with [35S]methionine and [3H]glucosamine. This glycoprotein was separated from phosphoglycan by chromatography on lentil lectin resin. The glycoprotein exhibited a L-tatrate-sensitive acid phosphatase activity, typical of secreted acid phosphatase (EC 3.1.3.2) from Leishmania. Monospecific antibodies to Leishmania donovani-secreted acid phosphatase selectively precipitated the L. tropica enzyme from immunoaffinity purified mixtures of the two antigens, and monoclonal antibodies to lipophosphoglycan precipitate the pure enzyme. Species-specific monoclonal antibodies to L. major lipophosphoglycan also recognized both L. tropica antigens. Treatment of the acid phosphatase with periodate or phosphodiesterase I abolished binding by the monoclonal antibodies to the pure enzyme. These results demonstrate that the two major secreted glycoconjugates of Leishmania tropica, the lipophosphoglycan and the acid phosphatase, share species-specific phosphorylated carbohydrate epitope(s). PMID:1697935

  4. Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

    Science.gov (United States)

    Xi, Jun; Yan, Huili

    2016-10-01

    Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies. PMID:27033966

  5. Applying generalized hydrophobicity scale of amino acids to quantitative prediction of human leukocyte antigen-A*0201-restricted cytotoxic T lymphocyte epitope

    Institute of Scientific and Technical Information of China (English)

    ZHOU Peng; TIAN Feifei; ZHANG Mengjun; LI Zhiliang

    2006-01-01

    Derived from 149 hydrophobic factors of 20 natural amino acids, a novel amino acid descriptor termed as generalized hydrophobicity scale (GH-scale) was proposed by principal component analysis (PCA). Via genetic algorithm-partial least square (GPLS) method, quantitative structure-activity relationship (QSAR) model was constructed by GH-scale for 152 human leukocyte antigen (HLA)-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes with the model estimated and cross-validated correlative coefficients of R2 = 0.813 and Q2 = 0.725, respectively. It was indicated that hydrophobic interaction played an important role in HLA-A*0201-CTL interaction, prominently at anchor residues.

  6. Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan. Partial characterization of private and public epitopes.

    Science.gov (United States)

    Ilg, T; Harbecke, D; Wiese, M; Overath, P

    1993-10-15

    -6[Glc beta 1,3]Gal beta 1,4Man alpha 1 repeats while six mAbs react with the unmodified repeats. Two antibodies specific for Leishmania major recognize Gal beta 1,3-substituted repeats unique for lipophosphoglycan from this species. Analysis by immunoblotting indicates that the high-molecular-mass proteo-phosphoglycan of L. mexicana secreted acid phosphatase carries epitopes for all anti-lipophosphoglycan mAbs suggesting the presence of capped phosphosaccharide repeats while the enzymically active glycoprotein subunit is modified by caps but probably not by repeats. In the case of Leishmania donovani secreted acid phosphatase, the enzymically active polypeptide may be directly modified by repeats. The mAbs are used to characterize changes in lipophosphoglycan structure, which occur in culture during the transition of promastigotes from the logarithmic to the stationary growth phase.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7693464

  7. Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

    Science.gov (United States)

    Riemer, D; Dodemont, H; Weber, K

    1991-12-01

    We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA. PMID:1724961

  8. Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2

    Directory of Open Access Journals (Sweden)

    Liu Chang M

    2011-08-01

    Full Text Available Abstract Background Porcine circovirus type 2 (PCV2 is associated with post-weaning multisystemic wasting syndrome (PMWS in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2. Results One monoclonal antibody (mAb; 8E4, against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2 strains but not PCV2b (YJ, SH and JF strains by an immunoperoxidase monolayer assay (IPMA and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2 strains but not PCV2b (YJ, SH and JF strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R in the capsid protein of PCV2a (CL, LG and JF2 strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4. Conclusions It is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2 strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2 strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease.

  9. Selection and Identification of Critical Amino Acids in Epitope 187-202 ofPen a1%Pen a1抗原表位187-202关键氨基酸的筛选和鉴定

    Institute of Scientific and Technical Information of China (English)

    牟慧; 高美须; 潘家荣; 支玉香; 赵杰; 刘超超; 李树锦; 赵鑫

    2014-01-01

    Objective Pen a1 is the major allergen in shrimp, and the sensitization mechanism is related with epitopes. The epitope (187-202) of Pen a1 was chosen as the research material, the amino acids’ frequency of occurrence and their conservative property were analyzed, the mutants were synthesized and the IgE capacity of the mutants were analyzed. The critical amino acids of the epitope of Pen a1 were screened out in order to study the shrimp sensitization mechanism and provide a theoretical basis for desensitization.[Method] The amino acid composition and frequency of occurrence in Pen a1, all epitopes and the studied epitope were analyzed using MEGA5, and the amino acids with the highest frequency were chosen. And then the amino acids of tropomyosin in all the allergenic foods in SDAP were also investigated, and the high conservative amino acids were chosen. The common amino acids in the results of both methods were considered to be the potential critical amino acids. Then these amino acids were substituted by alanine, the wild-type peptide and the mutant peptides were then synthesized with solid phase synthesis method. The tested serum was prepared by immuning New Zealand rabbits with wild-type peptide. The capacity of IgE-binding between the wild-type peptide and the mutants were compared with iELISA and competitive Dot-blot methods. The mutant peptides whose capacity of IgE-binding showed a dramatic decline were selected, and the replaced amino acids of the peptides were recognized as the critical amino acids.[Result] The amino acids of glutamic acid (E), leucine (L), arginine (R), glutamine (Q), valine (V), serine (S), aspartic acid (D) were found with higher frequency occurrence in epitope than inPen a1, and were considered as the active amino acids. E, V, L in epitope (187-202) were found with higher frequency occurrence, and were inferred to be the potential critical amino acids of studied epitope. The multiple sequence alignment of tropomyosin from

  10. Identification of critical amino acids in the IgE epitopes of Ric c 1 and Ric c 3 and the application of glutamic acid as an IgE blocker.

    Directory of Open Access Journals (Sweden)

    Natalia Deus-de-Oliveira

    Full Text Available BACKGROUND: The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients with glutamic acid reduced the IgE-epitope interaction. CONCLUSIONS/SIGNIFICANCE: The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.

  11. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

    Directory of Open Access Journals (Sweden)

    Peijnenburg Ad ACM

    2002-12-01

    Full Text Available Abstract Background Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase and allergenic proteins could be identified as (part of potential linear epitopes. Conclusion Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity.

  12. 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.

  13. Antigen epitope of Helicobacter pylorivacuolating cytotoxin A

    Institute of Scientific and Technical Information of China (English)

    Xiu-Li Liu; Shu-Qin Li; Chun-Jie Liu; Hao-Xia Tao; Zhao-Shan Zhang

    2004-01-01

    AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori) infection.METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA, cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro.RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20%and 30.41% respectively.CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

  14. Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

    Institute of Scientific and Technical Information of China (English)

    LIU Zuqiang; WANG Zuguang; CHEN Yinghua

    2005-01-01

    Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.

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

    DEFF Research Database (Denmark)

    Ozkokmen, D; Birkelund, Svend; Christiansen, Gunna

    1994-01-01

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

  16. Identification of critical amino acids in an immunodominant IgE epitope of Pen c 13, a major allergen from Penicillium citrinum.

    Directory of Open Access Journals (Sweden)

    Jui-Chieh Chen

    Full Text Available BACKGROUND: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A(148-E(166 and S22 (A(243-K(274 were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T(261-K(274, exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity. CONCLUSIONS/SIGNIFICANCE: Experimental analyses confirmed in silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic

  17. Epitope prediction methods

    DEFF Research Database (Denmark)

    Karosiene, Edita

    leucocyte antigen (HLA) molecules, are encoded by extremely polymorphic genes on chromosome 6. Due to this polymorphism, thousands of different MHC molecules exist, making the experimental identification of peptide-MHC interactions a very costly procedure. This has primed the need for in silico peptide......-MHC prediction methods, and over the last decade several such methods have been successfully developed and used for epitope discovery purposes. My PhD project has been dedicated to improve methods for predicting peptide-MHC interactions by developing new strategies for training prediction algorithms based...... 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...

  18. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6.

    Science.gov (United States)

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

    2016-08-01

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

  19. Identification of new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from neuritin

    OpenAIRE

    Yang , Zhao; Zhao, Tianzhi; Liu, Yong; Gong, Zili; Cheng, Saiyu; Yang, Qingwu

    2013-01-01

    Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. Neuritin, a recently discovered antigen overexpressed in astrocytoma, is considered to be a promising target for biological therapy. In the present study, we predicted and identified HLA-A2-restricted CTL epitopes from neuritin by using the following four-step procedure: (1) computer-based epitope prediction from the amino acid ...

  20. Dominant epitopes and allergic cross-reactivity

    DEFF Research Database (Denmark)

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

    2000-01-01

    as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order......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...... leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16...

  1. Immune epitope database analysis resource

    DEFF Research Database (Denmark)

    Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang;

    2012-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  3. Strategic Use of Epitope Matching to Improve Outcomes.

    Science.gov (United States)

    Wiebe, Chris; Nickerson, Peter

    2016-10-01

    Understanding the events leading to allorecognition and the subsequent effector pathways engaged is key for the development of strategies to prolong graft survival. Optimizing patient outcomes will require 2 major advancements: (1) minimizing premature death with a functioning graft in the patients with stable graft function, and (2) maximizing graft survival by avoiding the aforementioned allorecognition. This necessitates personalized immunosuppression to avoid known metabolic side effects, risk for infection, and malignancy, while holding the alloimmune system in check. Since the beginning of transplant a key strategy to achieve this goal is to minimize HLA mismatching between donor and recipient. What has not evolved is any refinement in our evaluation of HLA relatedness between donor and recipient when HLA mismatch exists. Donor-recipient HLA mismatch at the amino acid level can now be determined. These mismatches serve as potential epitopes for de novo donor specific antibody development and correlate with late rejection and graft loss. It is in this context that HLA epitope analysis is considered as a strategy to permit safe immunosuppression minimization to improve patient outcomes through: (1) improved allocation schemes that favor donor-recipient pairs with a low HLA epitope mismatch load (especially at the class II loci) or avoiding specific epitope mismatches known to be highly immunogenic and (2) immunosuppressive minimization in patients with low epitope mismatch loads or without highly immunogenic epitope mismatches.

  4. Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

    Science.gov (United States)

    Duquesnoy, R J; Hönger, G; Hösli, I; Marrari, M; Schaub, S

    2016-08-01

    The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry. PMID:27312793

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

    Science.gov (United States)

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

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

  6. Mapping of T cell epitopes using recombinant antigens and synthetic peptides.

    OpenAIRE

    Lamb, J R; Ivanyi, J.; Rees, A D; Rothbard, J B; Howland, K; Young, R. A.; Young, D B

    1987-01-01

    Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six...

  7. 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....

  8. Immune recognition of citrullinated epitopes.

    Science.gov (United States)

    Nguyen, Hai; James, Eddie A

    2016-10-01

    Conversion of arginine into citrulline is a post-translational modification that is observed in normal physiological processes. However, abnormal citrullination can provoke autoimmunity by generating altered self-epitopes that are specifically targeted by autoantibodies and T cells. In this review we discuss the recognition of citrullinated antigens in human autoimmune diseases and the role that this modification plays in increasing antigenic diversity and circumventing tolerance mechanisms. Early published work demonstrated that citrullinated proteins are specifically targeted by autoantibodies in rheumatoid arthritis and that citrullinated peptides are more readily presented to T cells by arthritis-susceptible HLA class II 'shared epitope' proteins. Emerging data support the relevance of citrullinated epitopes in other autoimmune diseases, including type 1 diabetes and multiple sclerosis, whose susceptible HLA haplotypes also preferentially present citrullinated peptides. In these settings, autoimmune patients have been shown to have elevated responses to citrullinated epitopes derived from tissue-specific antigens. Contrasting evidence implicates autophagy or perforin and complement-mediated membrane attack as inducers of ectopic citrullination. In either case, the peptidyl deiminases responsible for citrullination are activated in response to inflammation or insult, providing a mechanistic link between this post-translational modification and interactions with the environment and infection. As such, it is likely that immune recognition of citrullinated epitopes also plays a role in pathogen clearance. Indeed, our recent data suggest that responses to citrullinated peptides facilitate recognition of novel influenza strains. Therefore, increased understanding of responses to citrullinated epitopes may provide important insights about the initiation of autoimmunity and recognition of heterologous viruses. PMID:27531825

  9. Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

    International Nuclear Information System (INIS)

    We have previously shown that avian reovirus (ARV) σA and σNS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on σA (I and II) and three epitopes (A, B, and C) on σNS. To further define the location of epitopes on σA and σNS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of σA and σNS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on σA was located at amino acid residues 340QWVMAGLVSAA350 and epitope B on σNS at amino acid residues 180MLDMVDGRP188. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of σA and σNS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete σNS but not with truncated σNS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on σNS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on σA was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the σNS with MAb 1F9. The σNS of ARV with ssRNA-binding activity could be blocked by monoclonal antibody 1F9. The epitope B on σNS is required for ss

  10. Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

    Institute of Scientific and Technical Information of China (English)

    蓝灿辉; 田海军; 陈应华

    2004-01-01

    A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope,and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity.GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope.All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies,9D8 and 2D7,could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays.In the flow cytometry analysis,the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide,which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane.However,in syncytium assays,none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion.The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes.The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

  11. Autoantibody recognition mechanisms of p53 epitopes

    Science.gov (United States)

    Phillips, J. C.

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Tegar A. P. Siregar

    2016-03-01

    conservation ofT and B cell epitope in NS3 protein among DENV-4 strains and four serotypes DENV of Indonesia strains.Methods: Research was held at the Department of Microbiology, Faculty of Medicine, UniversitasIndonesia, June 2013 to April 2014. NS3 amino acid sequence of DENV-4 081 strain was obtained afterNS3 gene of DENV-4 081 PCR products were sequenced. T and B cell epitopes of NS3 protein of DENV-4081 strain were analysed and compared to NS3 proteins of 124 DENV-4 strains around the world and fourserotypes of Indonesia strains. World strains were isolated from America (i.e. Venezuela, Colombia, etc.and Asia (i.e. China, Singapore, etc.. For the comparison, T and B cell epitope positions of NS3 proteinwere obtained from published report.Results: Eight positions of T cell epitopes and two positions of B cell epitopes of NS3 DENV-4 081 wereidentical and conserved to NS3 protein of 124 DENV-4 strains around the world. B cell epitope of NS3 DENV-4 081 protein at aa 537-544 was found identical and conserved to four serotypes DENV of Indonesia strains.Conclusion: This wide conservation of T and B epitopes in almost all DENV-4 strains around the worldand all serotypes of Indonesia strains. (Health Science Journal of Indonesia 2015;6:126-31Keywords: dengue virus, NS3 protein, T cell epitope, B cell epitope

  13. GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders.

    Science.gov (United States)

    Fouka, P; Alexopoulos, H; Akrivou, S; Trohatou, O; Politis, P K; Dalakas, M C

    2015-04-15

    Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GAD-specific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes.

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

    DEFF Research Database (Denmark)

    Birkelund, Svend; Mygind, P; Holm, A;

    1996-01-01

    this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding...... to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay...... with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized...

  15. 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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzym...... that charged amino acids are essential for antibody reactivity and that the vimentin antibody is dependent on side-chain interactions in combination with backbone interactions....

  17. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation

    DEFF Research Database (Denmark)

    Yusim, K.; Kesmir, Can; Gaschen, B.;

    2002-01-01

    The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences...... reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env......, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First...

  18. The Possible Role of Transplacentally-Acquired Antibodies to Infectious Agents, With Molecular Mimicry to Nervous System Sialic Acid Epitopes, as Causes of Neuromental Disorders: Prevention and Vaccine Implications

    Directory of Open Access Journals (Sweden)

    André J. Nahmias

    2006-01-01

    Full Text Available Proof of causality of most neuromental disorders (NMD's is largely unavailable. Lessons from four-decade investigations of the epidemiology, immunology, pathogenesis, prevention and therapy of perinatal infectious agents, which invade directly the nervous system, have led us to propose a new indirect effect hypothesis: maternal transplacentally-acquired antibodies, to agents with epitope molecular mimicry with the developing nervous system, can cross the fetus/infant's blood–nervous system barriers to cause NMD's, clinically manifest years later.

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Induction of multi-epitope specific antibodies against HIV-1 by multi-epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strains in vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutation in vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutafions.

  1. A Novel Algorithm for Macromolecular Epitope Matching

    Directory of Open Access Journals (Sweden)

    Stanislav Jakuschev

    2009-03-01

    Full Text Available Many macromolecules, namely proteins, show functional substructures or epitopes defined by characteristic spatial arrangements of groups of specific atoms or residues. The identification of such substructures in a set of macromolecular 3D-structures solves an important problem in molecular biology as it allows the assignment of functions to molecular moieties and thus opens the possibility of a mechanistic understanding of molecular function. We have devised an algorithm that models a functional epitope formed by a group of atoms or residues as set of points in cartesian space with associated functional properties. The algorithm searches for similar epitopes in a database of structures by an efficient multistage comparison of distance sets in the epitope and in the structures from the database. The search results in a list of optimal matches and corresponding optimal superpositions of query epitope and matching epitopes from the database. The algorithm is discussed against the background of related approaches, and it is successfully tested in three application scenarios: global match of two homologous proteins, search for an epitope on a homologous protein, and finding matching epitopes in a protein database.

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

    Directory of Open Access Journals (Sweden)

    Yan-Ping Tian

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

  3. [Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity].

    Science.gov (United States)

    Tian, Wei-Wei; Qiao, Zhen-Hua; Yang, Lin-Hua; Wang, Hong-Wei; Tang, Yan-Hong; Bian, Si-Cheng

    2011-04-01

    This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.

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

    Directory of Open Access Journals (Sweden)

    Collier Kathy

    2002-01-01

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

  5. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer.

    Science.gov (United States)

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.

  6. Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1

    DEFF Research Database (Denmark)

    Holm, Jens; Ferreras, Mercedes; Ipsen, Henrik;

    2011-01-01

    allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction......Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen...

  7. The Immune Epitope Database 2.0

    DEFF Research Database (Denmark)

    Hoof, Ilka; Vita, R; Zarebski, L;

    2010-01-01

    of 4 years, the data from 180,978 experiments were curated manually from the literature, which covers approximately 99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise...... be unavailable to the public from 129,186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among...

  8. 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.

  9. The Use of the Immune Epitope Database to Study Autoimmune Epitope Data Related to Alopecia Areata.

    Science.gov (United States)

    Sette, Alessandro; Paul, Sinu; Vaughan, Kerrie; Peters, Bjoern

    2015-11-01

    The Immune Epitope Database (IEDB) is a repository of published epitope data for infectious diseases, allergy, transplantation and autoimmunity. Herein we provide an introduction to the IEDB search interface, focusing on data related to autoimmune diseases, including alopecia areata (AA). We demonstrate how common questions related can be answered, such as how to search for specific autoantigens, epitope sequences, response types (B- and/or T-cell assays), or host, as well as how to search for epitopes of known major histocompatibility complex restriction and for data related to a specific disease. Our survey of the data found that while as a whole Autoimmunity-specific records represent a significant portion (∼30%); epitopes reported for AA are remarkably few, just 23 epitopes from six antigens. This reveals a significant knowledge gap for AA, and suggests that additional mapping of epitopes and identification of novel AA-associated autoantigens is warranted. Citing recently published examples, we show how bioinformatic, proteomic, and technological advances make it now increasingly feasible to identify epitopes and novel antigens in human disease. The goal herein was to increase awareness of the IEDB as a free resource for the scientific community and to demonstrate its use in finding (existing) and analyzing (prediction) epitope data. PMID:26551944

  10. Rational Design of a Multiepitope Vaccine Encoding T-Lymphocyte Epitopes for Treatment of Chronic Hepatitis B Virus Infections▿

    OpenAIRE

    Depla, Erik; Der Aa, Annegret Van; Livingston, Brian D.; Crimi, Claire; Allosery, Koen; De Brabandere, Veronique; Krakover, Jonathan; Murthy, Sidharta; Huang, Manley; Power, Scott; Babé, Lilia; Dahlberg, Carol; McKinney, Denise; Sette, Alessandro; Southwood, Scott

    2007-01-01

    Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptid...

  11. Automatic Generation of Validated Specific Epitope Sets

    Directory of Open Access Journals (Sweden)

    Sebastian Carrasco Pro

    2015-01-01

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

  12. Synthetic Peptide libraries for T-cell epitope identification.

    Science.gov (United States)

    Hiemstra, H S; Drijfhout, J W; Koning, F

    2000-01-01

    This chapter describes a methodology for elucidating immunogenic epitopes stimulatory for CD4(+) T-cell clones (Fig. 1). The methodology makes use of synthetic peptide libraries and must be regarded as an alternative to other approaches, such as peptide elution or the application of genetic libraries. The methodology only requires knowledge about the restriction element of the T-cell clone. The restriction element determines which major histocompatibility complex (MHC)-binding anchor motif must be built into the library peptides. A synthetic peptide library is prepared comprising approx 8 million peptides. The synthesis proceeds via a mix-and-split protocol using a solidphase approach on a hybrid resin (1,2). On a hybrid resin, most of the peptide material (84%) is attached via an acid-labile linker whereas the remaining part of the peptide material is acid-stable attached (3). During synthesis, resinbound peptides comprising 14 amino acid residues are produced, with each resin bead containing one unique peptide (4,5). The beads are split into 384 pools, with each pool containing 20,000 beads. From each pool, about 28% of the peptide material is cleaved from every bead. Subsequently, in the first screening round, the 384 pools, each containing 20,000 solubilized peptides, are tested in a proliferation assay with the T-cell clone. Fig. 1. Flow diagram of the complete procedure for the identification of T-cell epitopes using synthetic peptide libraries (1).

  13. The epitope recognized by a monoclonal antibody in the myelin-associated protein CNP.

    Science.gov (United States)

    Stricker, R; Kalbacher, H; Reiser, G

    1997-08-18

    The epitope recognized by a monoclonal antibody (MAb-46-1) directed against the myelin-associated protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase; EC 3.1.4.37) from several species was characterized. MAb-46-1 can be employed for immunoprecipitation, immunostaining in Western blots and in immunohistochemistry. Short peptides derived from the human CNP1 peptide sequence were synthesized and used in enzyme linked immunosorbent assays to test the reactivity of MAb-46-1. Coarse screening experiments enabled us to localize the epitope recognized by MAb-46-1 to the amino acid residues 9 to 19 close to the N-terminus. Further investigations using shorter peptides comprising this part of the protein allowed us to identify a 9 amino acid residue long peptide (amino acids 11 to 19: ELQFPFLQD) which represents the minimal epitope recognized by MAb-46-1, probably through a 3-dimensional structure and less likely a straight linear peptide. The epitope seems to be stabilized also by the attached amino acids 7 to 10 (KDKP). The peptide sequence 9-19 is conserved in all CNP sequences described so far. Thus, MAb-46-1 might be of general usefulness for further studies of the not yet identified function of the myelin-associated protein CNP. PMID:9268698

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

    DEFF Research Database (Denmark)

    Adorini, L; Sette, A; Buus, S;

    1988-01-01

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

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

    Science.gov (United States)

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

    2015-11-27

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

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

    Science.gov (United States)

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

    2015-11-27

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71. PMID:26529072

  17. A highly conserved epitope-vaccine candidate against varicella-zoster virus induces neutralizing antibodies in mice.

    Science.gov (United States)

    Zhu, Rui; Liu, Jian; Chen, Chunye; Ye, Xiangzhong; Xu, Longfa; Wang, Wei; Zhao, Qinjian; Zhu, Hua; Cheng, Tong; Xia, Ningshao

    2016-03-18

    Varicella-zoster virus (VZV) is a highly infectious agent of varicella and herpes zoster (HZ). Vaccination is by far the most effective way to prevent these diseases. More safe, stable and efficient vaccines, such as epitope-based vaccines, now have been increasingly investigated by many researchers. However, only a few VZV neutralizing epitopes have been identified to date. We have previously identified a linear epitope between amino acid residues 121 and 135 of gE. In this study, we validated that this epitope is highly conserved amongst different VZV strains that covered five existing phylogenetic clades with an identity of 100%. We evaluated the immunogenicity of the recombinant hepatitis B virus core (HBc) virus-like particles (VLPs) which included amino acids (121-135). VZV-gE-specific antibodies were detected in immunized mouse serum using ELISA. The anti-peptide antiserum positively detected VZV via Western blot and immunofluorescent staining assays. More importantly, these peptides could neutralize VZV, indicating that these peptides represented neutralizing epitopes. These findings have important implications for the development of epitope-based protective VZV vaccines. PMID:26873057

  18. Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

    Directory of Open Access Journals (Sweden)

    Hanna Berkner

    Full Text Available Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Norcoclaurine synthase (NCS from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

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

    Directory of Open Access Journals (Sweden)

    Encheng Sun

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

  20. Expression and immunoreactivity of an epitope of HCV in a foreign epitope presenting system

    Institute of Scientific and Technical Information of China (English)

    Mei Peng; Chang-Bai Dai; Yuan-Ding Chen

    2005-01-01

    AIM: To construct and highly express an epitope of hepatitis C virus (HCV) in a foreign epitope presenting vectorbased on an insect virus, and to study the antigenicity of the epitope.METHODS: The HCV epitope sequence (amino acidresidues 315 to 328: EGHRMAWDMMMNWS) of the E1 region was constructed at different positions of a foreign epitope presenting vector based on an insect virus, flock house virus (FHV) capsid protein encoding gene as a vector, and expressed in E. coli cells. Western blottingand ELISA were used to detect the immunoreactivity of these recombinant proteins.RESULTS: The gene encoding of the concerned B-cell epitope of HCV E1 envelope protein was expressed on FHV capsid carrier protein at positions I1 (aa 106), I2 (aa153) and I3 (aa 305), respectively, on the surface of FHV capsid protein. The recombinant proteins in this system could be highly expressed in more than 40% of total cell protein of E. coli BL21. All the expressed recombinant proteins were in inclusion body form, and showed obvious immunoreactivity by Western blotting. Further purified recombinant proteins were detected by indirect ELISA as coating antigen respectively. All recombinant proteins could still show immunoreactivity.CONCLUSION: The epitope of HCV E1 envelope protein can be highly expressed in FHV carrier system as a chimeric protein with high immunoreactivity. This system has multiple entry sites conferring many possible conformations closer to the native one for a given sequence.

  1. Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein.

    Science.gov (United States)

    Thompson, Nancy E; Arthur, Terrance M; Burgess, Richard R

    2003-12-15

    Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the beta' subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins. PMID:14656522

  2. Characterization of T cell epitopes in bovine α-lactalbumin

    NARCIS (Netherlands)

    Meulenbroek, Laura A P M; den Hartog Jager, Constance F; Lebens, Ans F M; Knulst, André C; Bruijnzeel-Koomen, Carla A F M; Garssen, Johan; Knippels, Léon M J; van Hoffen, Els

    2014-01-01

    BACKGROUND: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this st

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

    Science.gov (United States)

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

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint. PMID:26715528

  4. In silico quantitative prediction of B-cell epitope

    Directory of Open Access Journals (Sweden)

    Raúl Isea

    2015-11-01

    Full Text Available This paper shows a computational approach for quantitative prediction of B cell epitopes. The function was defined, which reflects the average value of B epitopes, according to eight predictors of different B epitopes, as well as structural and energetic considerations of the origin protein. The proposed methodology could be useful to develop both dengue and chikungunya vaccines

  5. In silico quantitative prediction of B-cell epitope

    OpenAIRE

    Raúl Isea

    2015-01-01

    This paper shows a computational approach for quantitative prediction of B cell epitopes. The function was defined, which reflects the average value of B epitopes, according to eight predictors of different B epitopes, as well as structural and energetic considerations of the origin protein. The proposed methodology could be useful to develop both dengue and chikungunya vaccines

  6. Mapping of epitopes recognized by antibodies induced by immunization of mice with PspA and PspC.

    Science.gov (United States)

    Vadesilho, Cintia F M; Ferreira, Daniela M; Gordon, Stephen B; Briles, David E; Moreno, Adriana T; Oliveira, Maria Leonor S; Ho, Paulo L; Miyaji, Eliane N

    2014-07-01

    Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci. PMID:24807052

  7. Rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Sebastian Hoppe

    Full Text Available Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is

  8. Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

    Science.gov (United States)

    Hoppe, Sebastian; Bier, Frank F.; Nickisch-Rosenegk, Markus v.

    2013-01-01

    Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify

  9. Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

    Science.gov (United States)

    Nilsen, B M; Haugan, I R; Berg, K; Olsen, L; Brown, P O; Helland, D E

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN. PMID:8627677

  10. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

    Science.gov (United States)

    Yang, Yonglin; Lin, Shaoli; Nan, Yuchen; Ma, Zexu; Yang, Liping; Zhang, Yanjin

    2016-01-01

    Hepatitis E virus (HEV) is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3) encodes a small multifunctional protein (VP13), which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP) motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology. PMID:27548202

  11. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

    Directory of Open Access Journals (Sweden)

    Yonglin Yang

    2016-08-01

    Full Text Available Hepatitis E virus (HEV is one of the viral pathogens causing hepatitis in humans. HEV open reading frame 3 (ORF3 encodes a small multifunctional protein (VP13, which is essential for HEV infection. In this study, a linear epitope was identified in a polyproline (PXXP motif from VP13 of genotype 1 HEV by using a monoclonal antibody. The epitope was detected in enzyme-linked immunosorbent assay (ELISA, immunoblotting and immunofluorescence assays. Epitope mapping showed that the epitope locates in a proline-rich region containing a PXXP motif in amino acid residues 66-75 of VP13. The epitope was also detected in HEV-infected liver cells and reacted with genotype 1-specific antibodies in an HEV-positive human serum sample. The results demonstrated that the epitope in the PXXP motif of the genotype 1 VP13 is linear and surface-oriented, which should facilitate in-depth studies on the viral protein and HEV biology.

  12. Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines

    Directory of Open Access Journals (Sweden)

    Fusseder Nicolas

    2007-09-01

    Full Text Available Abstract Background In an epitope-based vaccine setting, the use of conserved epitopes would be expected to provide broader protection across multiple strains, or even species, than epitopes derived from highly variable genome regions. Conversely, in a diagnostic and disease monitoring setting, epitopes that are specific to a given pathogen strain, for example, can be used to monitor responses to that particular infectious strain. In both cases, concrete information pertaining to the degree of conservancy of the epitope(s considered is crucial. Results To assist in the selection of epitopes with the desired degree of conservation, we have developed a new tool to determine the variability of epitopes within a given set of protein sequences. The tool was implemented as a component of the Immune Epitope Database and Analysis Resources (IEDB, and is directly accessible at http://tools.immuneepitope.org/tools/conservancy. Conclusion An epitope conservancy analysis tool was developed to analyze the variability or conservation of epitopes. The tool is user friendly, and is expected to aid in the design of epitope-based vaccines and diagnostics.

  13. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  14. IgE-binding epitopes: a reappraisal

    NARCIS (Netherlands)

    R.C. Aalberse; R. Crameri

    2011-01-01

    Here, we discuss various questions related to IgE epitopes: What are the technical possibilities and pitfalls, what is currently known, how can we put this information into hypothetical frameworks and the unavoidable question: how useful is this information for patient care or allergenicity predicti

  15. Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

    DEFF Research Database (Denmark)

    Kroghsbo, Stine; Andersen, Nanna Birch; Rasmussen, Tina Frid;

    2014-01-01

    Background Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis. Objectives To examine...

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

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    Full Text Available BACKGROUND: 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. METHODS: 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. FINDINGS: 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. CONCLUSIONS: 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

  17. Detection and Quantification of CD4+ T Cells with Specificity for a New Major Histocompatibility Complex Class II-Restricted Influenza A Virus Matrix Protein Epitope in Peripheral Blood of Influenza Patients

    OpenAIRE

    Linnemann, Thomas; Jung, Günther; Walden, Peter

    2000-01-01

    FVFTLTVPS was identified as the core sequence of a new major histocompatibility complex class II-restricted T-cell epitope of influenza virus matrix protein. Epitope-specific CD4+ T cells were detected in the peripheral blood of patients with frequencies of up to 0.94%, depending on the number of additional terminal amino acids.

  18. Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity.

    Science.gov (United States)

    Debbink, Kari; Donaldson, Eric F; Lindesmith, Lisa C; Baric, Ralph S

    2012-01-01

    Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.

  19. Expression and immunoreactivity of HCV/HBV epitopes

    Institute of Scientific and Technical Information of China (English)

    Xin-Yu Xiong; Xiao Liu; Yuan-Ding Chen

    2005-01-01

    AIM: To develop the epitope-based vaccines to prevent Hepatitis C virus (HCV)/Hepatitis B virus (HBV) infections.METHODS: The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD (residuals aa2-21) and C2 VKFPGGGQIVGGVYLLPRR (residuals aa22-40), envelope epitope E GHRMAWDMMMNWSP (residuals aa315-328) and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC (residuals aa124-147) were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E. coli cells (pET-3 system).Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined.RESULTS: The expressed chimeric protein carrying the HCV epitopes C1, C2, E (two times), L3C1-I2E-L1C2-L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with antiHCV and anti-HBs antibodies.CONCLUSION: These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. HCV 抗原表位预测%Prediction of HCV antigenic epitopes

    Institute of Scientific and Technical Information of China (English)

    贾帅争; 孙红琰; 王全立

    2001-01-01

    应用网络生物信息资源查找丙型肝炎病毒基因组全序列,用软件Lasergene中的EditSeq将来自中国河北株mRNA序列翻译为氨基酸序列,尔后用程序Protean进行氨基酸序列分析,对HCV各区段的B细胞抗原指数进行预测。同时又在两个网站对中国汉族人中频率较高的HLA基因型进行CD8和CD4 T细胞表位预测。B细胞和T细胞抗原表位预测结果对于HCV诊断试剂和疫苗研制有重要的指导意义。%The complete genome of Hepatitis C China virus was gotten from world wide web site NCBI (National Center for Biotechnology Information). HCV mRNA was translated into amino acids(AA) sequence by program EditSeq of a computer software Lasergene and this amino acids sequence was analysed by program Protean. B cell epitopes index of these HCV AA fragments was caculated by this computational program and those epitopes with high index can be used as candidate epitopes for HCV antibody diagnositic reagent. CD8 T cell and CD4 T cell epitopes were predicted at Internet sites (SYFPEITHI and BIMAS). Because those HLA which appear with higher frequence in Chinese Nationalities were chosen to predict T cell epitopes, these predicted sequences can be used to design anti HCV vaccine suitable for Chinese.

  2. 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

    2016-02-01

    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. PMID:26514066

  3. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    Science.gov (United States)

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens.

  4. Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

    Directory of Open Access Journals (Sweden)

    Sapp Martin

    2006-10-01

    Full Text Available Abstract Background Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs, type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33 with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. Results Reactivities of monoclonal antibodies (mAbs H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa 51–58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132–140 and FGb (aa 282–291, were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260–270, in addition to loops DE and FGb. Conclusion These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.

  5. Identification of new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from neuritin.

    Science.gov (United States)

    Yang, Zhao; Zhao, Tianzhi; Liu, Yong; Gong, Zili; Cheng, Saiyu; Yang, Qingwu

    2013-08-01

    Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. Neuritin, a recently discovered antigen overexpressed in astrocytoma, is considered to be a promising target for biological therapy. In the present study, we predicted and identified HLA-A2-restricted CTL epitopes from neuritin by using the following four-step procedure: (1) computer-based epitope prediction from the amino acid sequence of neuritin; (2) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (3) stimulation of primary T cell response against the predicted peptides in vitro; and (4) testing of the induced CTLs toward target cells expressing neuritin and HLA-A2.1. The results demonstrated that effectors induced by peptides of neuritin containing residues 13-21, 121-129 and 4-12 could specifically-secrete interferon-γ and lyse target cells. Our results indicate that these peptides are new HLA-A2.1-restricted CTL epitopes, and may serve as valuable tools for astrocytoma immunotherapy. PMID:23754640

  6. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    Energy Technology Data Exchange (ETDEWEB)

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O' Neil, Karyn; Gilliland, Gary L.; (Centocor)

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  7. Prediction on Antigenic Epitope Characteristics of Bt Cry2Ab Protein in Transgenic Crops

    Institute of Scientific and Technical Information of China (English)

    Jierong GAO; Ying HE; Zehong ZOU; Ailin TAO; Yuncan AI

    2012-01-01

    Abstract [Objective] This study aimed to predict the structural characteristics of Bt Cry2Ab protein in transgenic crops with bioinformatic analysis to provide the theoreti- cal clues for design of antibody Cry2Ab. [Method] The amino acid sequence of Cry2Ab protein was searched from NCBI database. The B cell epitopes were pre- dicted with DNAStar. The binding affinity between Cry2Ab protein and MHC-II molecules was analyzed with NetMHCII 2.2 Server to predict the T cell epitopes. [Result] Prediction result suggested the potential B cell epitope of Cry2Ab locating in the region of 208-215. Analysis of the binding affinity between Cry2Ab and MHC-II molecules suggested the regions of 177-185, 299-307 and 255-263 were the po- tential T cell epitopes. Human with HLA-DRB10101 alleles and HLA-DRB10701 al- leles were more sensitive to Cry2Ab protein. [Conclusion] This study facilitates to un- derstand the structural characteristics of Cry2Ab protein and provides a new clue to improve the assessment method for potential allergenicity of genetically modified food.

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

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar

    2007-09-01

    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 crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  9. Identification of T. gondii epitopes, adjuvants, & host genetic factors that influence protection of mice & humans

    OpenAIRE

    Tan, Tze Guan; Mui, Ernest; Cong, Hua; Witola, William; Montpetit, Alexandre; Muench, Stephen P.; Sidney, John; Alexander, Jeff; Sette, Alessandro; Grigg, Michael; Maewal, Ajesh; McLeod, Rima

    2010-01-01

    Toxoplasma gondii is an intracellular parasite that causes severe neurologic and ocular disease in immune-compromised and congenitally infected individuals. There is no vaccine protective against human toxoplasmosis. Herein, immunization of Ld mice with HF10 (HPGSVNEFDF) with palmitic acid moieties or a monophosphoryl lipid A derivative elicited potent IFN-γ production from Ld-restricted CD8+ T cells in vitro and protected mice. CD8+ T cell peptide epitopes from T. gondii dense granule protei...

  10. Epitope peptides of influenza H3N2 virus neuraminidase gene designed by immunoinformatics

    Institute of Scientific and Technical Information of China (English)

    Lijun Liang; Ping Huang; Miaoheng Wen; Hanzhong Ni; Songnuan Tan; Yonghui Zhang; Qiuxia Chen

    2012-01-01

    The virus surface protein neuraminidase (NA) is a main subtype-specific antigen in influenza type A viruses.Neuraminidase functions as an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells.In this study,NA genes from the H3N2 subtype virus were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics.Based on this information,three peptides ES8,RR9,and WK7 (covering amino acid residues 221-228,292-300,and 383-389,respectively) of the NA protein were selected and synthesized artificially.These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera.Results showed that these three peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive manner,detected in vitro by enzyme-linked immunosorbent assay. Furthermore,hemadsorption anti-releasing effects occurred in three antisera mixtures at a dilution of 1∶40.Alignment using database software showed that amino acid residues in these three epitope peptides were substituted at specific sites in all the NAs sequenced in this study.We suggest that these NA epitope peptides might be used in conjunction with HA proteins as vaccine antigens.

  11. Development of wheat varieties with reduced contents of celiac-immunogenic epitopes through conventional and GM strategies

    NARCIS (Netherlands)

    Smulders, M.J.M.; Jouanin, A.A.; Schaart, J.G.; Visser, R.G.F.; Cockram, J.; Leigh, F.; Wallington, E.; Boyd, L.A.; Broeck, van den H.C.; Meer, van der I.M.; Gilissen, L.J.W.J.

    2014-01-01

    Cereals, especially wheat, may cause several food-related diseases, of which gluten intolerance (coeliac disease, CD) is the best defined: specific immunogenic epitopes, nine amino acid-long peptide sequences, have been identified from various gluten proteins. These may activate T cells, causing inf

  12. 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.

  13. Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Simon Rayner; Kang-hong Hu

    2011-01-01

    In recent years, the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein, a general overview of different tools currently available, including T cell and B cell epitopes prediction tools, is presented. And the principles of different prediction algorithms are reviewed briefly. Finally, several examples are present to illustrate the application of the prediction tools.

  14. Multiple HLA Epitopes Contribute to Type 1 Diabetes Susceptibility

    OpenAIRE

    Roark, Christina L.; Anderson, Kirsten M.; Simon, Lucas J.; Schuyler, Ronald P.; Aubrey, Michael T; Freed, Brian M.

    2013-01-01

    Disease susceptibility for type 1 diabetes is strongly associated with the inheritance of specific HLA alleles. However, conventional allele frequency analysis can miss HLA associations because many alleles are rare. In addition, disparate alleles that have similar peptide-binding sites, or shared epitopes, can be missed. To identify the HLA shared epitopes associated with diabetes, we analyzed high-resolution genotyping for class I and class II loci. The HLA epitopes most strongly associated...

  15. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    Science.gov (United States)

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG). PMID:27439498

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

    Directory of Open Access Journals (Sweden)

    Sylvain Cardinaud

    2011-05-01

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

  17. Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of Staphylococcus aureus Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding

    OpenAIRE

    Huesca, Mario; Sun, Qing; Peralta, Robert; Shivji, Gulnar M.; Sauder, Daniel N.; McGavin, Martin J.

    2000-01-01

    A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibo...

  18. SELECTION OF NEW EPITOPES FROM MONOVALENT DISPLAYED PHAGE OCTAPEPTIDE LIBRARY

    Institute of Scientific and Technical Information of China (English)

    李全喜; 王琰; 李竞; 王雅明; 徐建军; 王力民; 董志伟

    1998-01-01

    A library of 2×l07 random oetspaptides was constructed by use of phegemid-based monovaient phage display system. The randomly synthesized degenerated oilgodeoxyribonucleotides (oligos) were fused to the truncated gⅢ (p210-p408). Sequeraze analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (an) sequences are randomly distributed. The library was used to select binding paptides to the morroeloncl antlhody (mAb) 9E10, which recognizes a continuous decapaptide epitope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequerlce analysis of the selected positive clones indlcated that the binding sequences could fall into two chsses, one class (clone 1) shares a consensus motif, ISE x x L, with c-mire decapeprider and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically lnhlhited by froe c-myc deeapeptide. The immunogenlcitF cff the phage peptide was further investigsted h5, construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either done could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected psptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.

  19. Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

    Directory of Open Access Journals (Sweden)

    Pinghua Li

    Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

  20. Structure modeling and spatial epitope analysis for HA protein of the novel H1N1 influenza virus

    Institute of Scientific and Technical Information of China (English)

    WU Di; XU TianLei; SUN Jing; DAI JianXin; DING GuoHui; HE YunGang; ZHOU ZhengFeng; XIONG Hui; DONG Hui; JIN WeiRong; BIAN Chao; JIN Li; WANG HongYan; WANG XiaoNing; YANG Zhong; ZHONG Yang; WANG Hao; CHE XiaoYan; HUANG Zhong; LAN Ke; SUN Bing; WU Fan; YUAN ZhenAn; ZHANG Xi; ZHOU XiaoNong; ZHOU JiaHai; MA ZhiYong; TONG GuangZhi; GUO YaJun; ZHAO GuoPing; LI YiXue; CAO ZhiWei

    2009-01-01

    In recent months,a novel influenza virus H1N1 broke out around the world.With bioinformatics technology,the 3D structure of HA protein was obtained,and the epitope residues were predicted with the method developed in our group for this novel flu virus.58 amino acids were identified as potential epitope residues,the majority of which clustered at the surface of the globular head of HA protein.Although it is located at the similar position,the epitope of HA protein for the novel H1N1 flu virus has obvious differences in the electrostatic potential compared to that of HA proteins from previous flu viruses.

  1. Design and evaluation of a multi-epitope assembly Peptide (MEAP against herpes simplex virus type 2 infection in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Pan Mingjie

    2011-05-01

    Full Text Available Abstract Background Human herpes simplex virus (HSV 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2. Methods The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN from envelope glycoprotein B, 216-223 (GRTDRPSA from C, 6-18 (DPSLKMADPNRFR from D, 483-491 (DPPERPDSP from E, 572-579 (EPPDDDDS from G and 286-295 (CRRRYRRPRG from I glycoprotein of HSV-2, four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL from D, 162-177 (KDVTVSQVWFGHRYSQ from B, 205-224 (KAYQQGVTVDSIGMLPRFIP from D and 245-259 (KPPYTSTLLPPELSD from D and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK from D and 268-276 (ALLEDPAGT from D, are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290 of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3, and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. Results The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell

  2. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

    Institute of Scientific and Technical Information of China (English)

    Wang QM; Sun SH; Hu ZL; Zhou FJ; Yin M; Xiao CJ; Zhang JC

    2005-01-01

    Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.

  3. The molecular relationship between antigenic domains and epitopes on hCG.

    Science.gov (United States)

    Berger, Peter; Lapthorn, Adrian J

    2016-08-01

    Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGβ subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500Å(2) of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGβ (hCGβCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000Å(2) of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (Ł1+3) protruding from the central ck, encompassing h

  4. The molecular relationship between antigenic domains and epitopes on hCG.

    Science.gov (United States)

    Berger, Peter; Lapthorn, Adrian J

    2016-08-01

    Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGβ subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500Å(2) of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGβ (hCGβCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000Å(2) of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (Ł1+3) protruding from the central ck, encompassing h

  5. Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis.

    Science.gov (United States)

    Brandt, L; Oettinger, T; Holm, A; Andersen, A B; Andersen, P

    1996-10-15

    The recall of long-lived immunity in a mouse model of tuberculosis (TB) is defined as an accelerated accumulation of reactive T cells in the target organs. We have recently identified Ag 85B and a 6-kilodalton early secretory antigenic target, designated ESAT-6, as key antigenic targets recognized by these cells. In the present study, preferential recognition of the ESAT-6 Ag during the recall of immunity was found to be shared by five of six genetically different strains of mice. Overlapping peptides spanning the sequence of ESAT-6 were used to map two T cell epitopes on this molecule. One epitope recognized in the context of H-2b,d was located in the N-terminal part of the molecule, whereas an epitope recognized in the context of H-2a,k covered amino acids 51 to 60. Shorter versions of the N-terminal epitope allowed the precise definition of a 13-amino acid core sequence recognized in the context of H-2b. The peptide covering the N-terminal epitope was immunogenic, and a T cell response with the same fine specificity as that induced during TB infection was generated by immunization with the peptide in IFA. In the C57BL/6j strain, this single epitope was recognized by an exceedingly high frequency of splenic T cells (approximately 1:1000), representing 25 to 35% of the total culture filtrate-reactive T cells recruited to the site of infection during the first phase of the recall response. These findings emphasize the relevance of this Ag in the immune response to TB and suggest that immunologic recognition in the first phase of infection is a highly restricted event dominated by a limited number of T cell clones. PMID:8871652

  6. 4种血清型登革病毒NS1蛋白序列及B细胞抗原表位特异性分析%Analysis on genome and amino acids sequence and B cell epitopes for 4 serotypes of dengue virus NS1 protein

    Institute of Scientific and Technical Information of China (English)

    陈艳佳; 熊建英; 朱利; 曹虹; 赵卫

    2013-01-01

    目的 比较4种血清型登革病毒NS1蛋白型特异性抗原表位基因序列及氨基酸序列之间的差异,为利用基因差异进行血清学分型及疫苗研究提供新的线索.方法 利用DNAstar数据包中的Editseq程序,从20株登革病毒分离株的全基因组序列中将NS1基因型特异性抗原表位序列截取出来,再用Clustal X软件进行多序列比对,进行同源性分析,找出型内最为保守的抗原表位序列.并将比对结果在120株登革病毒序列中进一步验证.结果 NS1蛋白36~45和71~85位氨基酸为型特异性抗原表位,高度保守,型内完全相同,型间不同.结论 NS1蛋白36~45位氨基酸可以作为登革病毒血清分型和研制亚单位疫苗的靶标.%OBJECTIVE To compare genome and amino acids sequences and possible B cell epitopes of 4 serotypes of dengue virus NS1 protein, to explore a new method of gene typing and provide new clues to vaccine research. METHODS Cut off the corresponding gene sequences of NS1 protein from the complete genome of 20 dengue virus isolates, then multisequencing was carried out to find the gene which was conservative within the same serotype and variant in the other serotypes. RESULTS Specific antigens of NS1 protein (36-45 and 71-85 amino acids) were conservative within the same serotype and variant in other serotypes. CONCLUSION Specific antigens of NS1 protein (36-45 amino acids) are the bases of dengue virus typing and targets of subunit vaccine development.

  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...

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

    DEFF Research Database (Denmark)

    Zhang, Qing; Wang, Peng; Kim, Yohan;

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Almut Scherer

    2006-08-01

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

  10. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    Science.gov (United States)

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

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

    Science.gov (United States)

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

    2016-01-01

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

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Kiener Tanja K

    2012-02-01

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

  15. Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection.

    Science.gov (United States)

    Zhao, Zhuo; Sun, He-Qiang; Wei, Shan-Shan; Li, Bin; Feng, Qiang; Zhu, Jiang; Zeng, Hao; Zou, Quan-Ming; Wu, Chao

    2015-01-01

    No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31-48, SEB133-150, and SEB193-210. Six B-cell immunodominant epitopes (amino acid residues 31-48, 97-114, 133-150, 193-210, 205-222, and 247-261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.

  16. The protective antibodies induced by a novel epitope of human TNF-alpha could suppress the development of collagen-induced arthritis.

    Directory of Open Access Journals (Sweden)

    Jie Dong

    Full Text Available Tumor necrosis factor alpha (TNF-alpha is a major inflammatory mediator that exhibits actions leading to tissue destruction and hampering recovery from damage. At present, two antibodies against human TNF-alpha (hTNF-alpha are available, which are widely used for the clinic treatment of certain inflammatory diseases. This work was undertaken to identify a novel functional epitope of hTNF-alpha. We performed screening peptide library against anti-hTNF-alpha antibodies, ELISA and competitive ELISA to obtain the epitope of hTNF-alpha. The key residues of the epitope were identified by means of combinatorial alanine scanning and site-specific mutagenesis. The N terminus (80-91 aa of hTNF-alpha proved to be a novel epitope (YG1. The two amino acids of YG1, proline and valine, were identified as the key residues, which were important for hTNF-alpha biological function. Furthermore, the function of the epitope was addressed on an animal model of collagen-induced arthritis (CIA. CIA could be suppressed in an animal model by prevaccination with the derivative peptides of YG1. The antibodies of YG1 could also inhibit the cytotoxicity of hTNF-alpha. These results demonstrate that YG1 is a novel epitope associated with the biological function of hTNF-alpha and the antibodies against YG1 can inhibit the development of CIA in animal model, so it would be a potential target of new therapeutic antibodies.

  17. Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

    OpenAIRE

    He, X S; Rivkina, M; Stocker, B A; Robinson, W S

    1994-01-01

    To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into fun...

  18. Identification of Autoantigen Epitopes in Alopecia Areata.

    Science.gov (United States)

    Wang, Eddy H C; Yu, Mei; Breitkopf, Trisia; Akhoundsadegh, Noushin; Wang, Xiaojie; Shi, Feng-Tao; Leung, Gigi; Dutz, Jan P; Shapiro, Jerry; McElwee, Kevin J

    2016-08-01

    Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA. PMID:27094591

  19. Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

    Science.gov (United States)

    Anugraha, G; Madhumathi, J; Prince, P R; Prita, P J Jeya; Khatri, V K; Amdare, N P; Reddy, M V R; Kaliraj, P

    2015-10-01

    Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate.

  20. Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

    Science.gov (United States)

    Anugraha, G; Madhumathi, J; Prince, P R; Prita, P J Jeya; Khatri, V K; Amdare, N P; Reddy, M V R; Kaliraj, P

    2015-10-01

    Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate. PMID:26179420

  1. Dissecting antibodies with regards to linear and conformational epitopes.

    Science.gov (United States)

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

    2015-01-01

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

  2. Dissecting antibodies with regards to linear and conformational epitopes.

    Directory of Open Access Journals (Sweden)

    Björn Forsström

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

  3. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    Science.gov (United States)

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

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

    Directory of Open Access Journals (Sweden)

    Shen-Ju Gou

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

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

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Liao

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

  6. 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.

  7. 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.

  8. A pathogen-specific epitope inserted into recombinant secretory immunoglobulin A is immunogenic by the oral route.

    Science.gov (United States)

    Corthésy, B; Kaufmann, M; Phalipon, A; Peitsch, M; Neutra, M R; Kraehenbuhl, J P

    1996-12-27

    Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses. PMID:8969237

  9. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

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

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

  10. Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach

    DEFF Research Database (Denmark)

    Nielsen, Morten; Lundegaard, Claus; Worning, Peder;

    2004-01-01

    binding peptides and to guiding the process of rational vaccine design. Results: We apply the motif sampler method to the complex problem of MHC class II binding. The input to the method is amino acid peptide sequences extracted from the public databases of SYFPEITHI and MHCPEP and known to bind......Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus...

  11. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. T cell epitope-based allergy vaccines.

    Science.gov (United States)

    Larché, Mark

    2011-01-01

    Specific immunotherapy (SIT) with extracts containing intact allergen molecules is clinically efficacious, but associated with frequent adverse events related to the allergic sensitization of the patient. As a result, treatment is initiated in an incremental dose fashion which ultimately achieves a plateau (maintenance dose) that may be continued for several years. Reduction of allergic adverse events may allow safer and more rapid treatment Thus, many groups have developed and evaluated strategies to reduce allergenicity whilst maintaining immunogenicity, the latter being required to achieve specific modulation of the immune response. Peptide immunotherapy can be used to target T and/or B cells in an antigen-specific manner. To date, only approaches that target T cells have been clinically evaluated. Short, synthetic peptides representing immunodominant T cell epitopes of major allergens are able to modulate allergen-specific T cell responses in the absence of IgE cross linking and activation of effector cells. Here we review clinical and mechanistic studies associated with peptide immunotherapy targeting allergy to cats or to bee venom. 

  14. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O;

    2011-01-01

    Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype...

  15. Identification of a conserved linear neutralizing epitope recognized by monoclonal antibody 9A9 against serotype A foot-and-mouth disease virus.

    Science.gov (United States)

    Liang, Weifeng; Zhou, Guohui; Liu, Wenming; Yang, Baolin; Li, Chaosi; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Yu, Li

    2016-10-01

    Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, a series of outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope have the potential to provide protective immunity against diverse subtypes of FMDV serotype A and to protect against future pandemics. In this study, we produced an A serotype FMDV-specific monoclonal antibody (MAb) against the viral capsid protein VP1, designated 9A9, that potently neutralized FMDV A/JLYS/CHA/2014 with a 50 % neutralization titer (NT50) of 4,096. GST-fusion proteins expressing truncated peptides of VP1 were subjected to Western blot analysis using MAb 9A9, and it was found that the peptide (143)RGDLGPLAARL(153) of VP1 was the minimal epitope for MAb 9A9 binding. Western blot analysis also revealed that the epitope peptide could be recognized by positive sera from serotype A FMDV-infected pigs and cattle. Subsequent alanine-scanning mutagenesis analysis revealed that residues Gly(147) and Leu(149) of the 9A9-recognized epitope are crucial for MAb 9A9 binding. Furthermore, under immunological pressure selected by MAb 9A9, a single amino acid residue replacement (L149P) occurred in a viral neutralization-escape mutant, which verified the location of a critical residue of this epitope at Leu(149). Importantly, the epitope (143)RGDLGPLAARL(153) was highly conserved among different topotypes of serotype A FMDV strains in sequence alignment analysis. Thus, the results of this study could have application potential in the development of epitope-based vaccines and a suitable MAb-based diagnostic method for detection of type A FMDV as well as quantitation of antibodies against FMDV serotype A. PMID:27422396

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

    Science.gov (United States)

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

    2015-12-18

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

  17. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves' Disease.

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2012-05-01

    Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus. PMID:22405880

  20. 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.

  1. Minor interspecies variations in the sequence of the gp53 TSL-1 antigen of Trichinella define species-specific immunodominant epitopes.

    Science.gov (United States)

    Perteguer, M J; Rodríguez, E; Romarís, F; Escalante, M; Bonay, P; Ubeira, F M; Gárate, M T

    2004-06-01

    Among the Trichinella TSL-1 antigens, whose antigenicity is generally due mainly to tyvelose-containing epitopes, gp53 is unusual in that its antigenicity is due mainly to protein epitopes. In the present study we mapped two of these epitopes, recognized by monoclonal antibodies (mAbs) that specifically recognize gp53 from all encysting Trichinella species (mAb US9), or gp53 from Trichinella spiralis alone (mAb US5). Based on previously published sequences of this glycoprotein [Mol. Biochem. Parasitol. 72 (1995) 253], in this study, we cloned the full gp53 cDNA from a new strain, Trichinella britovi (ISS 11; AN: ), and from another T. spiralis isolate (ISS 115; AN: ). The gp53 sequence comprised an ORF of 1239bp, coding for 412 amino acids, with 61 nucleotide differences (resulting in 38 residue changes) between the two species. Mapping of US5- and US9-recognized epitopes was undertaken through the construction and expression in the pGEX4T vector of truncated gp53 peptides, and by the construction of peptides derived from the antigenic regions. The epitope recognized by mAb US9 was a linear peptide of 8 residues, 33Met- 40Ser, located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 was a 47-amino acid sequence containing two alpha-helix regions flanked by random coils, 290Thr- 336Lys. Molecular modeling of these peptides seems to indicate that recognition of the US9 epitope depends on the presence of two available hydroxyl groups provided by one methionine and one serine on T. spiralis gp53 (not present on Trichinella pseudospiralis gp53). Additionally, the stability of the US5 epitope seems to depend on correct folding of the 47-amino acid sequence (only present in T. spiralis). The relevance of these findings for understanding the antigenic recognition of Trichinella TSL-1 antigens, and for further studies to investigate possible function(s) of gp53 in Trichinella, is discussed. PMID:15163539

  2. Association of variations of NAb 2F5 and 4E10 epitopes and disease progression in Chinese antiretroviral treatment-na(i)ve patients infected with HIV-1 clade B'

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-li; JIANG Yong-jun; SHANG Hong; HAN Xiao-xu; DAI Di; BAO Ming-jia; XU Dong-bing; ZHANG Zi-ning; WANG Ya-nan; ZHAO Min; Tristan Bice

    2010-01-01

    Background Studies on human immunodeficiency virus type 1 (HIV-1) vaccines have recently focused on targeting the conserved neutralizing epitopes 2F5 and 4E10, and hence it is important to understand the extent of mutations in these two viral epitopes. Here, we investigated the amino acid mutations in epitopes of 2F5 (ELDKWA, HIV-1 HXB2 env 662-667 aa) and 4E10 (NWFDIT, HIV-1 HXB2 env 671-676 aa) in the membrane proximal-external region of gp41 from clade B' HIV-1-infected individuals living in Henan province, China. We also examined the frequency of a mutation and its relation to disease progression.Methods A cohort of 54 treatment-na(i)ve HIV-1-infected individuals was recruited in this study, and 16 individuals were selected for a short-term longitudinal study on sequence evolution. The HIV-1 env gp41 gene was amplified, cloned, and sequenced, and predicted amino acid sequences were aligned for analysis.Results The mutations E662A and K665E on the 2F5 epitope and N671S and T676S on the 4E10 epitope were seen.Simultaneous RNA sequencing showed some discrepancies with proviral DNA sequences. In our longitudinal study,mutation levels of these two neutralizing epitopes were low but diverse and persistent. The frequencies of mutations within the 4E10 peptide NWFDIT in slow progressors were noticeably lower than those in AIDS patients (P <0.05).Conclusions Antigenic variation of the neutralizing epitopes 2F5 and 4E10 is limited in subtype B' infection, and that 4E10 peptide mutation is correlated with disease progression. Monitoring epitope mutations will offer useful data for development of the candidate 2F5-like and 4E10-like antibodies to prevent and treat AIDS.

  3. Phage displaying epitope of Candida albicans HSP90 and serodiagnosis

    Institute of Scientific and Technical Information of China (English)

    杨琼; 王丽; 卢大宁; 邢沈阳; 尹东; 朱筱娟

    2004-01-01

    @@ Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused an increase in the frequency of life-threatening systemic candidiasis.1 Studies by Matthews et al2 indicated HSP90 fragments are major targets for the immune system in infection due to C. albicans, and anti-epitope LKVIRK of HSP90 antibody is a serological marker for diagnosis of invasive candidiasis. Cloning and sequencing HSP90 antigen revealed that the linear epitope LKVIRK, localized near the C-terminus of the 47 kDa protein which circulates in the sera of patients with invasive candidiasis, as a heat-stable breakdown product of large more heat-labile antigen HSP90.2 In this study, epitope LKVIRK was displayed on the surface of phage fd to develop a new serological test for systemic candidiasis.

  4. Epitope-Specific Binder Design by Yeast Surface Display.

    Science.gov (United States)

    Mann, Jasdeep K; Park, Sheldon

    2015-01-01

    Yeast surface display is commonly used to engineer affinity and design novel molecular interaction. By alternating positive and negative selections, yeast display can be used to engineer binders that specifically interact with the target protein at a defined site. Epitope-specific binders can be useful as inhibitors if they bind the target molecule at functionally important sites. Therefore, an efficient method of engineering epitope specificity should help with the engineering of inhibitors. We describe the use of yeast surface display to design single domain monobodies that bind and inhibit the activity of the kinase Erk-2 by targeting a conserved surface patch involved in protein-protein interaction. The designed binders can be used to disrupt signaling in the cell and investigate Erk-2 function in vivo. The described protocol is general and can be used to design epitope-specific binders of an arbitrary protein. PMID:26060073

  5. 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

    -negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility......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...... for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection...

  6. Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients.

    Science.gov (United States)

    Cusi, Maria Grazia; Botta, Cirino; Pastina, Pierpaolo; Rossetti, Maria Grazia; Dreassi, Elena; Guidelli, Giacomo Maria; Fioravanti, Antonella; Martino, Elodia Claudia; Gandolfo, Claudia; Pagliuchi, Marco; Basile, Assunta; Carbone, Salvatore Francesco; Ricci, Veronica; Micheli, Lucia; Tassone, Pierfrancesco; Tagliaferri, Pierosandro; Pirtoli, Luigi; Correale, Pierpaolo

    2015-09-01

    Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials. PMID:26031574

  7. Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

    Directory of Open Access Journals (Sweden)

    Tetsuji Kamata

    Full Text Available Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.

  8. A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

    Science.gov (United States)

    Velkov, Tony; Yun, Bo; Schneider, Elena K; Azad, Mohammad A K; Dolezal, Olan; Morris, Faye C; Nation, Roger L; Wang, Jiping; Chen, Ke; Yu, Heidi H; Wang, Lv; Thompson, Philip E; Roberts, Kade D; Li, Jian

    2016-05-13

    Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships. PMID:27627202

  9. Carcinoembryonic antigen continuous epitopes determined by the spot method.

    Science.gov (United States)

    Solassol, I; Granier, C; Pèlegrin, A

    2001-01-01

    Carcinoembryonic antigen (CEA) is a heavily glycosylated tumor-associated protein with an N-A1-B1-A2-B2-A3-B3 domain structure. Circulating CEA immunoassays are used for monitoring digestive cancer patients, and radiolabeled anti-CEA monoclonal antibodies (MAb) are used for the diagnosis and therapy of CEA-positive tumors. The five major nonoverlapping epitopes (Gold 1-5) have been broadly correlated with the domain organization, but there is no precise localization of the epitopes at the sequence level. In an attempt to identify the peptide sequences corresponding to the five Gold epitopes on the CEA molecule, we prepared a set of 227 overlapping fifteen-mer peptides corresponding to the complete CEA sequence with the SPOT method. Using five high affinity MAbs directed against the five CEA Gold epitopes, we demonstrated that none of these epitopes could be mimicked by a fifteen-mer peptide sequence. However, using rabbit and goat anti-CEA sera, we identified six major continuous antigenic regions. All are included in the Ig-like domains of the CEA: two in the A1 domain (residues 120-134 and 153-164), one each in the A2 (329-337) and A3 domains (508-513), one at the junction between the A3 and B3 domains (553-561) and one in the B3 domain (565-573). A very homologous sequence (common residues VSPRL) was mapped in each of the three A domains. Thus, in terms of occurrence of continuous epitopes, the Ig-like domains A1, A2, A3 and B3 seem to be the most antigenic parts of CEA. These peptide sequences should be good candidates for the future development of site-specific anti-CEA MAbs. PMID:11275797

  10. Antibody protection reveals extended epitopes on the human TSH receptor.

    Directory of Open Access Journals (Sweden)

    Rauf Latif

    Full Text Available Stimulating, and some blocking, antibodies to the TSH receptor (TSHR have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD. However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

  11. Antibody protection reveals extended epitopes on the human TSH receptor.

    Science.gov (United States)

    Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A; Davies, Terry F

    2012-01-01

    Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. PMID:22957097

  12. Genogeography and Immune Epitope Characteristics of Hepatitis B Virus Genotype C Reveals Two Distinct Types: Asian and Papua-Pacific.

    Directory of Open Access Journals (Sweden)

    Meta Dewi Thedja

    Full Text Available Distribution of hepatitis B virus (HBV genotypes/subgenotypes is geographically and ethnologically specific. In the Indonesian archipelago, HBV genotype C (HBV/C is prevalent with high genome variability, reflected by the presence of 13 of currently existing 16 subgenotypes. We investigated the association between HBV/C molecular characteristics with host ethnicity and geographical distribution by examining various subgenotypes of HBV/C isolates from the Asia and Pacific region, with further analysis on the immune epitope characteristics of the core and surface proteins. Phylogenetic tree was constructed based on complete HBV/C genome sequences from Asia and Pacific region, and genetic distance between isolates was also examined. HBV/C surface and core immune epitopes were analyzed and grouped by comparing the amino acid residue characteristics and geographical origins. Based on phylogenetic tree and geographical origins of isolates, two major groups of HBV/C isolates--East-Southeast Asia and Papua-Pacific--were identified. Analysis of core and surface immune epitopes supported these findings with several amino acid substitutions distinguishing the East-Southeast Asia isolates from the Papua-Pacific isolates. A west-to-east gradient of HBsAg subtype distribution was observed with adrq+ prominent in the East and Southeast Asia and adrq- in the Pacific, with several adrq-indeterminate subtypes observed in Papua and Papua New Guinea (PNG. This study indicates that HBV/C isolates can be classified into two types, the Asian and the Papua-Pacific, based on the virus genome diversity, immune epitope characteristics, and geographical distribution, with Papua and PNG as the molecular evolutionary admixture region in the switching from adrq+ to adrq-.

  13. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

    Directory of Open Access Journals (Sweden)

    Lin Na-Sheng

    2007-09-01

    Full Text Available Abstract Background Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV, that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. Methods We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s of the capsid protein VP1 of foot-and-mouth disease virus (FMDV. The recombinant BaMV plasmid (pBVP1 was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164 of FMDV VP1. Results The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. Conclusion Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  9. File list: Oth.NoD.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Oth.Adl.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  15. File list: Oth.Liv.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Oth.Bld.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX718015,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.50.Epitope_tags.AllCell.bed ...

  17. File list: Oth.EmF.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255459,SRX255462,SRX255460,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.20.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Adl.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.20.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.10.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Gon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204898,SR...X204899 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.50.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Neu.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.05.Epitope_tags.AllCell.bed ...

  3. File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.50.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Bld.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX180156,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.05.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...995,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.50.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Kid.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX170376,SRX065542,SRX065543 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.50.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Lng.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Lung SRX119639,SRX...119641,SRX119640,SRX119642,SRX119638,SRX119637 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.20.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211371,SRX493939,SRX381289 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Gon.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153152,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.20.Epitope_tags.AllCell.bed ...

  13. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

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

    OpenAIRE

    Kramer, Victor G; Byrareddy, Siddappa N.

    2014-01-01

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

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.10.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX759284,SRX691794,SRX759286,SRX691798,SRX691797,SRX691795,SRX022866,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.50.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Brs.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.05.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Emb.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066247,SRX066245,SRX815531 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.50.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Bon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Epd.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Emb.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.05.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Kid.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644727,SRX644719,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.YSt.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...1370,SRX211371,SRX493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.05.Epitope_tags.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.05.Epitope_tags.AllCell.bed ...

  8. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

    Energy Technology Data Exchange (ETDEWEB)

    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast

  9. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine

    Directory of Open Access Journals (Sweden)

    Hamid Nawaz-Tipu

    2015-10-01

    Full Text Available The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1 protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine’s candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Threealleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens moreprevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  10. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine.

    Science.gov (United States)

    Nawaz-Tipu, Hamid

    2015-10-01

    The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1) protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA) class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine's candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Three alleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens more prevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of 50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Boonsathorn, Naphatsawan; Panthong, Sumolrat [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Koksunan, Sarawut [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya [National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Prachasupap, Apichai [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Sasaki, Tadahiro [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); Yasugi, Mayo [Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); Ono, Ken-ichiro [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Arai, Yasuha [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); and others

    2014-09-26

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

  13. Epitope imprinted polymer nanoparticles containing fluorescent quantum dots for specific recognition of human serum albumin

    International Nuclear Information System (INIS)

    Epitope imprinted polymer nanoparticles (EI-NPs) were prepared by one-pot polymerization of N-isopropylacrylamide in the presence of CdTe quantum dots and an epitope (consisting of amino acids 598 to 609) of human serum albumin (HSA). The resulting EI-NPs exhibit specific recognition ability and enable direct fluorescence quantification of HSA based on a fluorescence turn-on mode. The polymer was characterized by FT-IR, X-ray photoelectron spectroscopy, transmission electron microscopy and dynamic light scattering. The linear calibration graph was obtained in the range of 0.25–5 μmol · mL−1 with the detection limit of 44.3 nmol · mL−1. The EI-NPs were successfully applied to the direct fluorometric quantification of HSA in samples of human serum. Overall, this approach provides a promising tool to design functional fluorescent materials with protein recognition capability and specific applications in proteomics. (author)

  14. Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain.

    Science.gov (United States)

    Cui, Z; Qin, A; Lee, L F; Wu, P; Kung, H J

    1999-01-01

    A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.

  15. Identification and Characterization of Peptides Mimicking the Epitopes of Metalloprotease of Schistosoma Japonicum

    Institute of Scientific and Technical Information of China (English)

    Lianfei Tang; Yuxiao Chen; Linqian Wang; Shunke Zhang; Xianfang Zeng; Xinyuan Yi

    2005-01-01

    In an attempt to isolate and characterize peptides mimicking epitopes of metalloprotease and explore their immunological protection against Schistosoma japonicum (S. japonicum), polyclonal anti-metalloprotease sera was prepared to screen a 12-mer random peptide library to isolate phages binding specially to antisera IgG. Then,phage ELISA, animal immunization, DNA sequencing, Western blotting and enzymatic activity neutralizing analysis were used to characterize the selected phage clones. All of ten randomly picked clones were shown to be positive. Five peptides of different amino acid sequences deduced from DNA sequences were obtained and two of them (peptides 2 and 3) could induce significant reduction (31.0% and 31.8%, respectively) in worm burden and high reduction (52.6% and 54.9%, respectively) in liver eggs per gram (LEPG), while, unexpectedly, others (peptides 1, 4 and 5) could not elicit enough protection against infection of S. japonicum. Peptides 2 and 3 could be recognized by S. japonicum infected mouse sera (IMS) and could elicit neutralizing Abs. The results show that peptides 2 and 3 are antigenic and immunogenic. They are true mimics of epitopes of metalloprotease and useful as novel vaccine candidates against S. japonicum.

  16. Epitope hunting in rheumatoid arthritis : towards antigen specific immunotherapy

    NARCIS (Netherlands)

    de Jong, H.

    2013-01-01

    Current treatment options in rheumatoid arthritis aim to dampen the immune response a-specifically. In the last decennia new strategies have emerged that have fewer side effects due to more specificity by focussing on those cells of the immune system that deal with regulation. Epitope specific immun

  17. An epitope delivery system for use with recombinant mycobacteria

    NARCIS (Netherlands)

    Hetzel, C.; Janssen, R.; Ely, S.J.; Kristensen, N.M.; Bunting, K.; Cooper, J.B.; Lamb, J.R.; Young, D.B.; Thole, J.E.R.

    1998-01-01

    We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- G

  18. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

    Science.gov (United States)

    Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

    2014-02-01

    Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine.

  19. An epitope tag derived from human transcription factor IIB that reacts with a polyol-responsive monoclonal antibody.

    Science.gov (United States)

    Duellman, Sarah J; Thompson, Nancy E; Burgess, Richard R

    2004-05-01

    Polyol-responsive monoclonal antibodies (PR-mAbs) provide a strategy to purify active, nondenatured proteins by a single-step immunoaffinity chromatography procedure. The high affinity interaction between these antibodies and the antigen can be dissociated in the presence of a nonchaotropic salt and a low molecular weight polyhydroxylated compound (polyol). The epitope for PR-mAb IIB8 is located near the N-terminus of the human transcription factor IIB (TFIIB). The epitope is an eight amino acid sequence, TKDPSRVG, that can be fused to a desired protein for use as a purification tag. This epitope tag (termed hIIB) was fused to the C-terminus of green fluorescent protein (GFP). An additional GFP fusion protein utilized another version of hIIB containing a point mutation at position two. These fusion proteins, expressed in Escherichia coli, allowed successful separation of the desired protein in a single chromatographic step. This strategy extends PR-mAb gentle-release purification to numerous expressed proteins. PMID:15039078

  20. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

    Science.gov (United States)

    Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

    2014-02-01

    Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine. PMID:24054942

  1. MIMOX: a web tool for phage display based epitope mapping

    Directory of Open Access Journals (Sweden)

    Honda Wataru

    2006-10-01

    Full Text Available Abstract Background Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. Results We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. Conclusion A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

  4. 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.

  5. 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. PMID:27223692

  6. Epitope-specific antibody levels in tuberculosis: biomarkers of protection, disease and response to treatment.

    Directory of Open Access Journals (Sweden)

    Graham H Bothamley

    2014-06-01

    Full Text Available Monoclonal antibodies restricted to Mycobacterium tuberculosis can measure epitope-specific antibody levels in a competition assay. Immunodominant epitopes were defined from clinical samples and related to the clinical spectrum of disease. Antibody to the immunodominant epitopes was associated with HLA-DR15. Occupational exposure showed a different response and was consistent with recognition of dormancy related proteins and protection despite exposure to tuberculosis. Studies in leprosy revealed the importance of immune deviation and the relationships between T and B cell epitopes. During treatment, antibody levels increased, epitope spreading occurred, but the affinity constants remained the same after further antigen exposure, suggesting constraints on the process of epitope selection. Epitope-specific antibody levels have a potential role as biomarkers for new vaccines which might prevent the progression of latent to active tuberculosis and as tools to measure treatment effects on subpopulations of tubercle bacilli.

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

    International Nuclear Information System (INIS)

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

  8. OptMAVEn--a new framework for the de novo design of antibody variable region models targeting specific antigen epitopes.

    Directory of Open Access Journals (Sweden)

    Tong Li

    Full Text Available Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn. OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β2 m-free heavy chains.

    Science.gov (United States)

    Tremante, Elisa; Lo Monaco, Elisa; Ingegnere, Tiziano; Sampaoli, Camilla; Fraioli, Rocco; Giacomini, Patrizio

    2015-08-01

    Since HLA-E heavy chains accumulate free of their light β2 -microglobulin (β2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2 m-associated and β2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies. PMID:25982269

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

    Science.gov (United States)

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

    2012-09-21

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

  12. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

    Directory of Open Access Journals (Sweden)

    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  13. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    Science.gov (United States)

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  14. 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.

  15. Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.

    OpenAIRE

    Wu, J Y; Newton, S; Judd, A; Stocker, B; Robinson, W S

    1989-01-01

    A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and t...

  16. Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

    OpenAIRE

    Fu, T. M.; Bonneau, R H; Tevethia, M J; Tevethia, S S

    1993-01-01

    Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycopro...

  17. Three Candidate Epitope-Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV-1

    Institute of Scientific and Technical Information of China (English)

    刘祖强; 田海军; 王颖; 陈应华

    2003-01-01

    HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines.So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies.Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HIV-1.According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated E1, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2: C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits.After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies.An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide.Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine.Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response.This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 μg.Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.

  18. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The minigenes encoding Plasmodium falciparum CTL epitopesrestricted to human MHC class I molecular HLA-A2 and HLA-B51, which were both at high frequency among Chinese population, were constructed as mono-epitope CTL vaccines named pcDNA3.1/tr and pcDNA3.1/ sh. The minigenes of the two epitopes were then tandem linked to form a dimeric CTL epitope minigene recombinant vaccine. After DNA transfection, the epitope minigenes were expressed respectively in two human cell lines, each bearing one MHC class I molecule named CIR/HLA-A2.1 and K562/HLA-B51. The intracellular expression of the CTL epitope minigenes not only enhanced the stability of HLA-A2.1 and HLA-B51 molecules but also increased the assemblage of MHC class I molecules on cell surfaces, which testified the specific process and presentation of those endogenous expressed epitopes. For the cells transfected with the dimeric minigene encoding two tandem linked epitopes, the expression and presentation of each epitope were also detected on cell membranes that bore different MHC class I molecules. It meant that the adjacency of the two CTL epitopes did not interfere with the specific process and presentation of each epitope. Compared with the ordinary CTL studies that inoculated synthesized epitope peptides with peripheral blood cells, this work aimed to process the epitopes directly inside HLA class I allele specific human cells, and thus theoretically imitated the same procedure in vivo. It was also an economical way to predict the immunogenicity of CTL epitopes at an early stage especially in laboratories with limited financial resource.

  19. Identification of an epitope of SARS-coronavirus nucleocapsid protein

    Institute of Scientific and Technical Information of China (English)

    YING LIN; JIN WANG; HONG XIA WANG; HUA LIANG JIANG; JIAN HUA SHEN; YOU HUA XIE; YUAN WANG; GANG PEI; BEI FEN SHEN; JIA RUI WU; BING SUN; XU SHEN; RUI FU YANG; YI XUE LI; YONG YONG JI; YOU YU HE; MUDE SHI; WEI LU; TIE LIU SHI

    2003-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV werepredicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodieswere isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-inducedpolyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratorysyndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

  20. Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

    Institute of Scientific and Technical Information of China (English)

    徐沁; 丁建芳; 胡红雨; 许根俊

    1999-01-01

    The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.

  1. '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

  2. Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide.

    OpenAIRE

    Apicella, M A; Shero, M; Jarvis, G A; Griffiss, J. M.; Mandrell, R E; Schneider, H.

    1987-01-01

    Gonococcal lipooligosaccharides (LOSs) are a series of antigenically complex heteropolymers. To investigate whether all members of clonally selected populations of Neisseria gonorrhoeae express antigenically similar LOS, we studied gonococcal strains 4505 and 220 with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or comigrating LOS unit(s) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent-antibody and immun...

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

    Directory of Open Access Journals (Sweden)

    Joos Thomas

    2010-06-01

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

  4. Mapping epitopes and antigenicity by site-directed masking

    Science.gov (United States)

    Paus, Didrik; Winter, Greg

    2006-06-01

    Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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......The interaction between antibodies and antigens is one of the most important immune system mechanisms for clearing infectious organisms from the host. Antibodies bind to antigens at sites referred to as B-cell epitopes. Identification of the exact location of B-cell epitopes is essential in several...... 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...

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

    Science.gov (United States)

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

    2010-05-01

    Both linear and conformational epitopes likely contribute to the allergenicity of tree nut allergens, yet, due largely to technical issues, few conformational epitopes have been characterized. Using the well studied recombinant cashew allergen, Ana o 2, an 11S globulin or legumin, we identified a murine monoclonal antibody which recognizes a conformational epitope and competes with patient IgE Ana o 2-reactive antibodies. This epitope is expressed on the large subunit of Ana o 2, but only when associated with an 11S globulin small subunit. Both Ana o 2 and the homologous soybean Gly m 6 small subunits can foster epitope expression, even when the natural N-terminal to C-terminal subunit order is reversed in chimeric molecules. The epitope, which is also expressed on native Ana o 2, is readily susceptible to destruction by physical and chemical denaturants. PMID:20362336

  7. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    Science.gov (United States)

    Kowalsky, Caitlin A; Faber, Matthew S; Nath, Aritro; Dann, Hailey E; Kelly, Vince W; Liu, Li; Shanker, Purva; Wagner, Ellen K; Maynard, Jennifer A; Chan, Christina; Whitehead, Timothy A

    2015-10-30

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. PMID:26296891

  8. Identification of Cytotoxic T Lymphocyte Epitopes on Swine Viruses: Multi-Epitope Design for Universal T Cell Vaccine

    OpenAIRE

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

    2013-01-01

    Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8+ T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an ex...

  9. 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....... This experimental validation of computational predictions illustrates the potential for immunoinformatics methods to identify candidate immunogens for a new, safer smallpox vaccine....

  10. Conserved regions ofPlasmodium vivax potential vaccine candidate antigens in Sri Lanka:Consciousin silico analysis of prospective conformational epitope regions

    Institute of Scientific and Technical Information of China (English)

    Shanika Amarasinghe; Hashendra Kathriarachchi; Preethi Udagama

    2014-01-01

    Objective:To do mapping and modeling of conformationalB cell epitope regions of highly conserved and protective regions of three merozoitecandidate vaccine proteins ofPlasmodium vivax(P. vivax) ,ie. merozoite purface protein-1(PvMSP-1), apical membrane antigen -1 domainⅡ(PvAMA1-DⅡ) and regionⅡ of theDuffy binding protein(PvDBPⅡ), and to analyze the immunogenic properties of these predicted epitopes.Methods:3-D structures of amino acid haplotypes fromSriLanka(available inGeneBank) ofPvMSP-119(n=27),PvAMA1-DⅡ(n=21) andPvDBPⅡ(n=33) were modeled.SEPPA, selected as the best online server was used for conformational epitope predictions, while prediction and modeling of protein structure and properties related to immunogenicity was carried out withGeno3D server,SCRATCHProtein Server,NetSurfPServer and standalonesoftware,Genious5.4.4.Results:SEPPA revealed that regions of predicted conformational epitopes formed4 clusters inPvMSP-I19, and3 clusters each inPvAMA1-DⅡ andPvDBPⅡ, all of which displayed a high degree of hydrophilicity, contained solvent exposed residues, displayed high probability of antigenicity and showed positive antigenic propensity values, that indicated high degree of immunogenicity.Conclusions:Findings of this study revealed and confirmed that different parts of the sequences of each of the conserved regions of the three selected potential vaccine candidate antigens ofP. vivax are important with regard to conformational epitope prediction that warrants further laboratory experimental investigations in in vivo animal models.

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

    Directory of Open Access Journals (Sweden)

    Ting Chen

    2008-09-01

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

  12. Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction

    Directory of Open Access Journals (Sweden)

    Huang Jun

    2012-09-01

    Full Text Available Abstract Background Severe acute respiratory syndrome (SARS is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction. Results We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A, lysine (K or aspartic acid (D, respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously. Conclusions We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.

  13. Identification of Immunodominant Th1-type T cell Epitopes from Schistosoma japonicum 28 kDa Glutathione-S-transferase, a Vaccine Candidate

    Institute of Scientific and Technical Information of China (English)

    Guang-Fu LI; Guan-Ling WU; Yong WANG; Zhao-Song ZHANG; Xin-Jun WANG; Min-Jun JI; Xiang ZHU; Feng LIU; Xiao-Ping CAI; Hai-Wei WU

    2005-01-01

    Th1-type cytokines produced by the stimulation of Th1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection.Schistosoma mansoni 28 kDa glutathione-S-transferase (Sm28GST), a major detoxification enzyme, has been recognized as a vaccine candidate and a phase Ⅱ clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST (Sj28GST) have shown immune protection against the parasite infection. In the present study, six candidate peptides (P1, P2, P3, P4, P7 and P8) from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope (190-211 aa) identified from Sm28GST was selected and named P9.The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c(+)/BL21(DE3) system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6(73-86 aa) generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ and IL-2. Therefore, P6 is a functional Th1-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Th1-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.

  14. Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

    Institute of Scientific and Technical Information of China (English)

    Maria Homs; Maria Buti; David Tabernero; Josep Quer; Alex Sanchez; Noelia Corral; Rafael Esteban

    2012-01-01

    AIM:To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS).METHODS:Four samples (2 genotype A and 2 genotype D) from 4 treatment-naive patients were assessed for baseline variability.Two additional samples from one patient (patient 4,genotype D) were selected for analysis:one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment.The HBV region analyzed covered amino acids 40 to 95 of the core gene,and included the two main epitopic regions,Th50-69 and B74-84.UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences,Roche).After computer filtering of UDPS data based on a Poisson statistical model,122 813 sequences were analyzed.The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture.RESULTS:Positions with highest variability rates were mainly located in the main core epitopes,confirming their role as immune-stimulating regions.In addition,the distribution of variability showed a relationship with HBV genotype.Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%.Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%,respectively.The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed,approaching significance (P =0.07,sample 1 and P =0.05,sample 2).In contrast,there were no significant differences in variability between the epitopic and other positions in genotype D cases.Interestingly,patient 1 presented a completely mutated motif from amino acid 64 to 67 (E64LMT67),which is commonly recognized by T helper cells.Additionally,the variability observed in all 4 patients was particularly associated with the E64

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

    Directory of Open Access Journals (Sweden)

    Rok Seon Choung

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

  16. A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

    Science.gov (United States)

    Kim, Ji-Nu; Yi, Jeong Sang; Lee, Bo-Rahm; Kim, Eun-Jung; Kim, Min Woo; Song, Yoseb; Cho, Byung-Kwan; Kim, Byung-Gee

    2012-07-20

    Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor. PMID:22704935

  17. Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Myriam A. Herrera

    1994-01-01

    Full Text Available Multiple antigen peptide systems (MAPs allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30 from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11 and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25 proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

  18. Conformational epitope mapping of Pru du 6, a major allergen from almond nut.

    Science.gov (United States)

    Willison, LeAnna N; Zhang, Qian; Su, Mengna; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2013-10-01

    Tree nuts are a widely consumed food. Although enjoyed safely by most individuals, allergic reactions to tree nuts, including almond, are not uncommon. Almond prunin (Pru du 6), an 11S globulin (legumin), is an abundant nut seed protein and a major allergen. Conformational epitope mapping studies of prunin have been performed with a murine monoclonal antibody (mAb) 4C10. This mAb reacts with non-reduced but not reduced prunin in immunoblotting assays, indicating the recognition of a conformational epitope. 4C10 competes with patient IgE, as assessed by ELISA, indicating clinical significance of the epitope. To characterize the 4C10 epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (MS) was performed on the native prunin-4C10 complex and on uncomplexed native prunin. Several epitope candidate peptides that differ in deuterium uptake between the complexed and uncomplexed forms were identified. The epitope was further mapped by analyzing chimeric molecules incorporating segments of the homologous soybean allergen, Gly m 6, in immunoassays. These data indicate that the 4C10 epitope overlaps with a subset of patient IgE binding epitopes on almond prunin and further supports HDX-MS as a valid technique for mapping conformational epitopes. PMID:23498967

  19. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes

    DEFF Research Database (Denmark)

    Corbet, Sylvie; Nielsen, Henrik Vedel; Vinner, Lasse;

    2003-01-01

    more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes. This...... study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple...

  20. Epitopes associated with the MHC restriction site of T cells. II. Somatic generation of Iat epitopes on T cells in radiation bone marrow chimeras

    Energy Technology Data Exchange (ETDEWEB)

    Asano, Y.; Tada, T.

    1987-01-01

    We described in this paper systematic alterations in the expression of unique I region controlled epitopes on helper T cells (Th) in chimeras according to the changes in their H-2 restriction specificity. Taking advantage of the reactivity of monoclonal antibodies (anti-Iat) putatively specific for the epitopes indirectly controlled by I region and expressed in association with the Iak restriction site of Th, we examined the alterations of these epitopes on Th cells from various bone marrow chimeras. Iatk epitopes were physiologically expressed on Iak-restricted but not on Iab-restricted Th cells in (H-2k X H-2b)F1 mice. In the chimeric condition, the H-2k-restricted Th of B6----F1 chimera acquired the expression of Iatk even though B6 Th is unable to express Iatk when developed under the physiologic condition. Iatk are also found on Th of fully allogeneic chimera of B6----C3H, whereas Th cells of C3H----B6 completely lost the Iatk expression. These results indicate that Iat epitopes originally defined as unique I region-controlled determinants selectively expressed on T cells are not encoded by the I region genes but are associated with the T cell receptor that sees the self Ia. The epitopes undergo the adaptive alterations according to the acquisition of a new MHC restriction. This is the first example to demonstrate the epitope associated with T cell receptor which undergo the systematic adaptive differentiation.

  1. Epitopes associated with the MHC restriction site of T cells. II. Somatic generation of Iat epitopes on T cells in radiation bone marrow chimeras

    International Nuclear Information System (INIS)

    We described in this paper systematic alterations in the expression of unique I region controlled epitopes on helper T cells (Th) in chimeras according to the changes in their H-2 restriction specificity. Taking advantage of the reactivity of monoclonal antibodies (anti-Iat) putatively specific for the epitopes indirectly controlled by I region and expressed in association with the Iak restriction site of Th, we examined the alterations of these epitopes on Th cells from various bone marrow chimeras. Iatk epitopes were physiologically expressed on Iak-restricted but not on Iab-restricted Th cells in (H-2k X H-2b)F1 mice. In the chimeric condition, the H-2k-restricted Th of B6----F1 chimera acquired the expression of Iatk even though B6 Th is unable to express Iatk when developed under the physiologic condition. Iatk are also found on Th of fully allogeneic chimera of B6----C3H, whereas Th cells of C3H----B6 completely lost the Iatk expression. These results indicate that Iat epitopes originally defined as unique I region-controlled determinants selectively expressed on T cells are not encoded by the I region genes but are associated with the T cell receptor that sees the self Ia. The epitopes undergo the adaptive alterations according to the acquisition of a new MHC restriction. This is the first example to demonstrate the epitope associated with T cell receptor which undergo the systematic adaptive differentiation

  2. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1 epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes

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    Lee Sang-Guk

    2009-08-01

    Full Text Available Abstract Background To identify novel HLA-A*2402-restricted human cytomegalovirus (HCMV immediate early-1 (IE-1 epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. Methods These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-γ (IFN-γ using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. Results Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs producing IFN-γ. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-11–15MESSAKRKMDPDNPD and IE-15–19AKRKMDPDNPDEGPS induced the greatest quantities of IFN-γ production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402-restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-11–15 and IE-15–19. Peptide IE-13–12SSAKRKMDPD induced the greatest quantities of IFN-γ production and target cell killing by CD8+ CTLs. Conclusion HCMV IE-13–12SSAKRKMDPD is a HLA-A*2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.

  3. Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

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    Titti Fausto

    2004-09-01

    Full Text Available Abstract Background The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis, a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. Results A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii detection of ~42 and 84 kDa proteins by Western blot and (iii the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. Conclusion This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

  4. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2

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    Youngshin Han

    2016-05-01

    Full Text Available Ara h 2, a peanut 2S albumin, is associated with severe allergic reactions, but a homologous protein, soybean 2S albumin, is not recognized as an important allergen. Structural difference between these proteins might explain this clinical discrepancy. Therefore, we mapped sequential epitopes and compared the structure of Ara h 2, Soy Al 1, and Soy Al 3 (Gly m 8 to confirm whether structural differences account for the discrepancy in clinical responses to these two proteins. Commercially synthesized peptides covering the full length of Ara h 2 and two soybean 2S albumins were analyzed by peptide microarray. Sera from 10 patients with peanut and soybean allergies and seven non-atopic controls were examined. The majority of epitopes in Ara h 2 identified by microarray are consistent with those identified previously. Several regions in the 2S albumins are weakly recognized by individual sera from different patients. A comparison of allergenic epitopes on peanut and soybean proteins suggests that loop-helix type secondary structures and some amino acids with a large side chain including lone electron pair, such as arginine, glutamine, and tyrosine, makes the peptides highly recognizable by the immune system. By utilizing the peptide microarray assay, we mapped IgE epitopes of Ara h 2 and two soybean 2S albumins. The use of peptide microarray mapping and analysis of the epitope characteristics may provide critical information to access the allergenicity of food proteins.

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

    Institute of Scientific and Technical Information of China (English)

    GAO; Jun; GONG; Yuping; ZHAO; Ping; ZHU; Qing; YANG; Xiaoping; QI; Zhongtian

    2006-01-01

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

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

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    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.

  7. Characterisation and epitope analysis of monoclonal antibodies to virions of clover yellow vein and Johnsongrass mosaic potyviruses.

    Science.gov (United States)

    Hewish, D R; Xiao, X W; Mishra, A; Gough, K H; Shukla, D D

    1993-01-01

    Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (ClYVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of ClYVV a total of ten MAbs were obtained which reacted strongly with ClYVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five ClYVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with ClYVV, form the BYMV subgroup of potyvirses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The ClYVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five ClYVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the ClYVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of ClYVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the ClYVV coat protein. Comparison of published coat protein sequences of BYMV and ClYVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of ClYVV coat protein as these MAbs

  8. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens

    Science.gov (United States)

    Caro-Aguilar, Ivette; Rodríguez, Alexandra; Calvo-Calle, J. Mauricio; Guzmán, Fanny; De la Vega, Patricia; Elkin Patarroyo, Manuel; Galinski, Mary R.; Moreno, Alberto

    2002-01-01

    Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development. PMID:12065487

  9. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein.

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    Mark D Hicar

    Full Text Available Numerous broadly neutralizing antibodies (Abs target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes. Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs, we previously used HIV virus-like particles (VLPs to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.

  10. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein.

    Science.gov (United States)

    Hicar, Mark D; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U; Kalams, Spyros A; Doranz, Benjamin J; Spearman, Paul; Crowe, James E

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jo L. Chung

    2010-01-01

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

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

    DEFF Research Database (Denmark)

    Stranzl, Thomas

    CTL methods, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively. Part III reports the results of an analysis investigating how the alternatively spliced cancer exome differs from the exome of normal tissue in terms of containing predicted MHC class I binding....... Part IV of the thesis deals with the analysis of 93 patient-donor pairs that underwent hematopoietic stem cell transplantation (HCT). HCT is a standard treatment for a variety of hematological diseases. Graft-versus-host disease is a possible complication after an HCT, where the recipient´s cells...

  14. Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets.

    Science.gov (United States)

    Wu, Ko-Wen; Chien, Chih-Yi; Li, Shiao-Wen; King, Chwan-Chuen; Chang, Chuan-Hsiung

    2012-08-01

    This study focused on identifying the conserved epitopes in a single subtype A (H3N2)-as candidates for vaccine targets. We identified a total of 32 conserved epitopes in four viral proteins [22 HA, 4PB1, 3 NA, 3 NP]. Evaluation of conserved epitopes in coverage during 1968-2010 revealed that (1) 12 HA conserved epitopes were highly present in the circulating viruses; (2) the remaining 10 HA conserved epitopes appeared with lower percentage but a significantly increasing trend after 1989 [p<0.001]; and (3) the conserved epitopes in NA, NP and PB1 are also highly frequent in wild-type viruses. These conserved epitopes also covered an extremely high percentage of the 16 vaccine strains during the 42 year period. The identification of highly conserved epitopes using our approach can also be applied to develop broad-spectrum vaccines. PMID:22698979

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

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    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.

  16. Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1.

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    Lilian Lacerda Bueno

    Full Text Available 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.

  17. Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection.

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    Xiao Fang Lim

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is the main causative agent of Hand, Foot and Mouth disease (HFMD and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16. CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

  18. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

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    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

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

    Science.gov (United States)

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

    2016-02-01

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

  20. Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

    Institute of Scientific and Technical Information of China (English)

    Ping Li; Xiaodong Cui; Yuying Li; Zhuanhua Wang

    2011-01-01

    Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named Fl, F2, F3, and F4, respectively.Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb.According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3).Following purification by Ni2+ affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient).Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg139 and Asp144 might be involved in the allergic activity of TBb.However, R141A had the weakest IgE-binding activity,suggesting that Arg141 may be the critical amino acid of TBb.This is the first report on the epitope mapping and identification of TBb.Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.

  1. Plasmodium falciparum: an epitope within a highly conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen is a target of parasite-inhibitory antibodies.

    Science.gov (United States)

    Fox, B A; Xing-Li, P; Suzue, K; Horii, T; Bzik, D J

    1997-02-01

    Previously, the Plasmodium falciparum serine repeat antigen has been shown to be protective in primate models of malaria immunity and also to be a target of in vitro parasite-inhibitory antibodies. To further define parasite-inhibitory epitopes a series of deletions from the amino-terminal 47-kDa domain of the serine repeat antigen (SERA) were constructed as glutathione-S-transferase fusion proteins. Several GST-SERA fusion proteins were used to vaccinate mice with Freund's adjuvant and the resulting immune sera were used to assay for the inhibition of P. falciparum invasion of erythrocytes in vitro. The minimal epitope shown to be the target of invasion-blocking antibodies was SERA amino acids 17-165. Additional GST-SERA deletion constructs of the 47-kDa domain were developed and evaluated for reactivity, by Western immunoblot analysis, with a parasite-inhibitory murine monoclonal antibody (mAb 43E5), a parasite-inhibitory pooled goat polyclonal sera, and a pooled human Nigerian immune serum. The parasite-inhibitory epitope defined by mAb 43E5 was mapped to SERA amino acids 17-110 and, at least, part of the epitope was defined to include amino acids in the region of amino acids 59-72. The parasite-inhibitory epitope recognized by mAb 43E5 appears to be well conserved between diverse geographical isolates of P. falciparum. The results have relevance for malaria vaccine development and suggest that an appropriately designed recombinant SERA antigen produced from a synthetic gene in Escherichia coli may be an effective component of a candidate malaria vaccine. PMID:9030663

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  3. NetCTLpan: pan-specific MHC class I pathway epitope predictions

    DEFF Research Database (Denmark)

    Stranzl, Thomas; Larsen, Mette Voldby; Lundegaard, Claus;

    2010-01-01

    Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates...

  4. Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes.

    Directory of Open Access Journals (Sweden)

    Barbara Hjelm

    Full Text Available A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  5. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs. Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  6. HLA-DQ molecules as affinity matrix for identification of gluten T cell epitopes.

    Science.gov (United States)

    Dørum, Siri; Bodd, Michael; Fallang, Lars-Egil; Bergseng, Elin; Christophersen, Asbjørn; Johannesen, Marie K; Qiao, Shuo-Wang; Stamnaes, Jorunn; de Souza, Gustavo A; Sollid, Ludvig M

    2014-11-01

    Even though MHC class II is a dominant susceptibility factor for many diseases, culprit T cell epitopes presented by disease-associated MHC molecules remain largely elusive. T cells of celiac disease lesions recognize cereal gluten epitopes presented by the disease-associated HLA molecules DQ2.5, DQ2.2, or DQ8. Employing celiac disease and complex gluten Ag digests as a model, we tested the feasibility of using DQ2.5 and DQ2.2 as an affinity matrix for identification of disease-relevant T cell epitopes. Known gluten T cell epitope peptides were enriched by DQ2.5, whereas a different set of peptides was enriched by DQ2.2. Of 86 DQ2.2-enriched peptides, four core sequences dominated. One of these core sequences is a previously known epitope and two others are novel epitopes. The study provides insight into the selection of gluten epitopes by DQ2.2. Furthermore, the approach presented is relevant for epitope identification in other MHC class II-associated disorders. PMID:25261484

  7. IgE epitopes of intact and digested Ara h 1

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and fou...

  9. B-Cell Receptor Epitope Recognition Correlates With the Clinical Course of Chronic Lymphocytic Leukemia

    NARCIS (Netherlands)

    Binder, Mascha; Mueller, Fabian; Jackst, Antje; Lechenne, Barbara; Pantic, Milena; Bacher, Ulrike; Eulenburg, Christine Zu; Veelken, Hendrik; Mertelsmann, Roland; Pasqualini, Renata; Arap, Wadih; Trepel, Martin

    2011-01-01

    BACKGROUND: B-cell receptors (BCRs) and their recognition of specific epitopes may play a pivotal role in the development and progression of chronic lymphocytic leukemia (CLL). In this study, the authors set up a model system to explore epitope reactivity and its clinical relevance in CLL. METHODS:

  10. Molecular modeling and conformational IgG epitope mapping on bovine β-casein

    NARCIS (Netherlands)

    Liu, Fahui; Gao, Jinyan; Li, Xin; Chen, Hongbing

    2016-01-01

    Characterizing conformational B-cell epitopes is a key step for understanding the immunological basis of allergy contributed by β-casein. There is no resolved conformational structure of β-casein in protein data bank, and most of the previous research on epitope identification of β-casein focused

  11. T Cell Epitope Peptide Therapy for Allergic Diseases.

    Science.gov (United States)

    O'Hehir, Robyn E; Prickett, Sara R; Rolland, Jennifer M

    2016-02-01

    Careful selection of dominant T cell epitope peptides of major allergens that display degeneracy for binding to a wide array of MHC class II molecules allows induction of clinical and immunological tolerance to allergen in a refined treatment strategy. From the original concept of peptide-induced T cell anergy arising from in vitro studies, proof-of-concept murine models and flourishing human trials followed. Current randomized, double-blind, placebo-controlled clinical trials of mixtures of T cell-reactive short allergen peptides or long contiguous overlapping peptides are encouraging with intradermal administration into non-inflamed skin a preferred delivery. Definitive immunological mechanisms are yet to be resolved but specific anergy, Th2 cell deletion, immune deviation, and Treg induction seem implicated. Significant efficacy, particularly with short treatment courses, in a range of aeroallergen therapies (cat, house dust mite, grass pollen) with inconsequential non-systemic adverse events likely heralds a new class of therapeutic for allergy, Synthetic Peptide Immuno-Regulatory Epitopes (SPIRE). PMID:26768622

  12. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    Energy Technology Data Exchange (ETDEWEB)

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  13. High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.

    Science.gov (United States)

    Brooks, Benjamin D; Miles, Adam R; Abdiche, Yasmina N

    2014-08-01

    Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection. PMID:24880105

  14. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes

    DEFF Research Database (Denmark)

    Corbet, S; Nielsen, HV; Vinner, L;

    2003-01-01

    CTL epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic...

  15. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes

    DEFF Research Database (Denmark)

    Corbet, S.; Nielsen, H.V.; Vinner, L.;

    2003-01-01

    epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic and more...

  16. ElliPro: a new structure-based tool for the prediction of antibody epitopes

    Directory of Open Access Journals (Sweden)

    Fusseder Nicholas

    2008-12-01

    Full Text Available Abstract Background Reliable prediction of antibody, or B-cell, epitopes remains challenging yet highly desirable for the design of vaccines and immunodiagnostics. A correlation between antigenicity, solvent accessibility, and flexibility in proteins was demonstrated. Subsequently, Thornton and colleagues proposed a method for identifying continuous epitopes in the protein regions protruding from the protein's globular surface. The aim of this work was to implement that method as a web-tool and evaluate its performance on discontinuous epitopes known from the structures of antibody-protein complexes. Results Here we present ElliPro, a web-tool that implements Thornton's method and, together with a residue clustering algorithm, the MODELLER program and the Jmol viewer, allows the prediction and visualization of antibody epitopes in a given protein sequence or structure. ElliPro has been tested on a benchmark dataset of discontinuous epitopes inferred from 3D structures of antibody-protein complexes. In comparison with six other structure-based methods that can be used for epitope prediction, ElliPro performed the best and gave an AUC value of 0.732, when the most significant prediction was considered for each protein. Since the rank of the best prediction was at most in the top three for more than 70% of proteins and never exceeded five, ElliPro is considered a useful research tool for identifying antibody epitopes in protein antigens. ElliPro is available at http://tools.immuneepitope.org/tools/ElliPro. Conclusion The results from ElliPro suggest that further research on antibody epitopes considering more features that discriminate epitopes from non-epitopes may further improve predictions. As ElliPro is based on the geometrical properties of protein structure and does not require training, it might be more generally applied for predicting different types of protein-protein interactions.

  17. Epitope-driven DNA vaccine design employing immunoinformatics against B-cell lymphoma: a biotech's challenge.

    Science.gov (United States)

    Iurescia, Sandra; Fioretti, Daniela; Fazio, Vito Michele; Rinaldi, Monica

    2012-01-01

    DNA vaccination has been widely explored to develop new, alternative and efficient vaccines for cancer immunotherapy. DNA vaccines offer several benefits such as specific targeting, use of multiple genes to enhance immunity and reduced risk compared to conventional vaccines. Rapid developments in molecular biology and immunoinformatics enable rational design approaches. These technologies allow construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways, as well as highly targeted vaccines aimed at specific epitopes. Reliable predictions of immunogenic T cell epitope peptides are crucial for rational vaccine design and represent a key problem in immunoinformatics. Computational approaches have been developed to facilitate the process of epitope detection and show potential applications to the immunotherapeutic treatment of cancer. In this review a number of different epitope prediction methods are briefly illustrated and effective use of these resources to support experimental studies is described. Epitope-driven vaccine design employs these bioinformatics algorithms to identify potential targets of vaccines against cancer. In this paper the selection of T cell epitopes to develop epitope-based vaccines, the need for CD4(+) T cell help for improved vaccines and the assessment of vaccine performance against tumor are reviewed. We focused on two applications, namely prediction of novel T cell epitopes and epitope enhancement by sequence modification, and combined rationale design with bioinformatics for creation of new synthetic mini-genes. This review describes the development of epitope-based DNA vaccines and their antitumor effects in preclinical research against B-cell lymphoma, corroborating the usefulness of this platform as a potential tool for cancer therapy. Achievements in the field of DNA vaccines allow to overcome hurdles to clinical translation. In a scenario where the vaccine

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

    Directory of Open Access Journals (Sweden)

    Janet Lei

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

  19. Development of peptide-based lineage-specific serology for chronic Chagas disease: geographical and clinical distribution of epitope recognition.

    Directory of Open Access Journals (Sweden)

    Tapan Bhattacharyya

    2014-05-01

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

  20. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice.

    Science.gov (United States)

    Jafarpour, Nazli; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Kohram, Fatemeh; Aghababa, Haniyeh; Khoramabadi, Nima; Mahdavi, Mehdi

    2014-08-01

    Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine. PMID:24842263

  1. Epitope mapping of PfCP-2.9, an asexual blood-stage vaccine candidate of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    He Zhicheng

    2010-04-01

    Full Text Available Abstract Background Apical membrane antigen 1 (AMA-1 and merozoite surface protein 1 (MSP1 of Plasmodium falciparum are two leading blood-stage malaria vaccine candidates. A P. falciparum chimeric protein 2.9 (PfCP-2.9 has been constructed as a vaccine candidate, by fusing AMA-1 domain III (AMA-1 (III with a C-terminal 19 kDa fragment of MSP1 (MSP1-19 via a 28-mer peptide hinge. PfCP-2.9 was highly immunogenic in animal studies, and antibodies elicited by the PfCP-2.9 highly inhibited parasite growth in vitro. This study focused on locating the distribution of epitopes on PfCP-2.9. Methods A panel of anti-PfCP-2.9 monoclonal antibodies (mAbs were produced and their properties were examined by Western blot as well as in vitro growth inhibition assay (GIA. In addition, a series of PfCP-2.9 mutants containing single amino acid substitution were produced in Pichia pastoris. Interaction of the mAbs with the PfCP-2.9 mutants was measured by both Western blot and enzyme-linked immunosorbent assay (ELISA. Results Twelve mAbs recognizing PfCP-2.9 chimeric protein were produced. Of them, eight mAbs recognized conformational epitopes and six mAbs showed various levels of inhibitory activities on parasite growth in vitro. In addition, seventeen PfCP-2.9 mutants with single amino acid substitution were produced in Pichia pastoris for interaction with mAbs. Reduced binding of an inhibitory mAb (mAb7G, was observed in three mutants including M62 (Phe491→Ala, M82 (Glu511→Gln and M84 (Arg513→Lys, suggesting that these amino acid substitutions are critical to the epitope corresponding to mAb7G. The binding of two non-inhibitory mAbs (mAbG11.12 and mAbW9.10 was also reduced in the mutants of either M62 or M82. The substitution of Leu31 to Arg resulted in completely abolishing the binding of mAb1E1 (a blocking antibody to M176 mutant, suggesting that the Leu residue at this position plays a crucial role in the formation of the epitope. In addition, the Asn15

  2. A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes

    Directory of Open Access Journals (Sweden)

    Sue D. Xiang

    2015-10-01

    Full Text Available Sperm protein antigen 17 (Sp17, expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17 sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional “mix-in” pro-inflammatory adjuvant CpG, both mapping to amino acids (aa 111–142. However, delivery of hSp17111–142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111–142, from an immuno-dominant region 134–142 aa for CpG, to region 121–138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses.

  3. A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes.

    Science.gov (United States)

    Xiang, Sue D; Gao, Qian; Wilson, Kirsty L; Heyerick, Arne; Plebanski, Magdalena

    2015-01-01

    Sperm protein antigen 17 (Sp17), expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17) sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional "mix-in" pro-inflammatory adjuvant CpG, both mapping to amino acids (aa) 111-142. However, delivery of hSp17111-142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111-142, from an immuno-dominant region 134-142 aa for CpG, to region 121-138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses. PMID:26529027

  4. Prediction of B Cell Epitope of DMRT Protein in Oreochromis niloticus%尼罗罗非鱼DMRT蛋白 B细胞表位的预测

    Institute of Scientific and Technical Information of China (English)

    杨东; 刘红艳; 张繁荣; 余来宁

    2008-01-01

    [Objective] The research aimed to predict the B cell epitope of DMRT protein in Oreochromis niloticus. [Method] The secondary structure of amino acid sequence of DMRT protein was revealed by Garnier-Robson, Chou-Fasman and Karplus-Schulz methods. The hydrophilicity plot, surface probability and antigenic index were obtained by Kyte-Doolittle, Emini and Jameson-Wolf methods, respectively. Based on the above results, the B cell epitopes for DMRT were predicted. [Result] Both the prediction results from Garnier-Robson, Chou-Fasman methods indicated that the α-helix centers of DMRT protein in O. niloticus were in the N terminal No. 31-56, 68-75, 110-116, 209-211 and 239-243; the β-sheet centers of DMRT protein in O. niloticus were in the N terminal No. 95-99, 177-183, 225-234 and 251-254. With the assistant of Kyte-Doolittle, Emini and Jameson-Wolf methods, the B cell epitopes for DMRT were located in or nearby the N terminal No. 13-16, 35-38, 47-54,84-93, 101-109, 127-156, 166-177 and 198-201. [Conclusion] These results are helpful for preparing the antibody of DMRT protein and revealing the sex determination mechanism of O. niloticus.

  5. 一种实用的筛选病毒抗原表位方法的建立%An Applicable Method for Mapping Epitopes on Viral Protein

    Institute of Scientific and Technical Information of China (English)

    孙涛; 陆苹; 王欣

    2004-01-01

    采用基因分段克隆、表达结合蛋白质印迹, 筛选到了口蹄疫病毒非结构蛋白3ABC上高结合力、保守的感染相关线性表位,分别位于3ABC蛋白上第106~155和156~190位氨基酸.这两个表位可与感染不同血清型口蹄疫病毒动物康复血清反应,但不与来自健康免疫动物和未接触病毒动物的血清发生反应.实验表明,用基因工程表达的多肽筛选抗原表位的方法是可行的.%By the means of overlapping peptides expressed in Escherichia coli in combination with Western blotting, two linear epitopes were identified on non-structural protein 3ABC of foot-and-mouth disease virus(FMDV), which covering amino acid residues 106~155 and 156~190 on 3ABC. The epitopes gave positive reaction with sera from pigs or guinea pigs infected with different serotypes of FMDV, but not with sera from vaccinated or naive animals. The method based on expressed peptide for mapping epitope on viral protein was reliable and applicable.

  6. Elicitation of neutralizing antibodies directed against CD4-induced epitope(s using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Antu K Dey

    Full Text Available The identification of HIV-1 envelope glycoprotein (Env structures that can generate broadly neutralizing antibodies (BNAbs is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4 receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i epitope(s known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH, was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140 using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1 complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s. These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s here, and its potential role in vaccine application.

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

    Science.gov (United States)

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

    2016-02-01

    Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines. PMID:26489873

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

    Directory of Open Access Journals (Sweden)

    Wenhan Chen

    Full Text Available Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/

  9. Multipin peptide libraries for antibody and receptor epitope screening and characterization.

    Science.gov (United States)

    Tribbick, Gordon

    2002-09-01

    It has been nearly 15 years since the papers describing the fully systematic epitope mapping approach both for the so-called "continuous" epitopes [Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 3998] and "discontinuous" epitopes [Mol. Immunol. 23 (1986) 709] were published. These seminal papers laid the conceptual foundation for all subsequent developments where a combinatorial approach is applied. Dr. Mario Geysen, the 2000 Kilby Laureate, can certainly lay claim to be the "father of combinatorial chemistry" (http://www.kilby.org/laureates.htm). In this review, I will focus on the aspects of the Multipin technology as they apply to antibody and receptor epitope mapping. Much of what will be presented applies equally well to other applications where peptide libraries (PepSets) and combinatorial approaches are used [Rodda, S.J., 1996. T-cell epitope mapping with synthetic peptides and peripheral blood mononuclear cells. In: Morris, G.E. (Eds.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, Chap. 30, p. 363; Int. J. Pept. Protein Res. 42 (1993) 384; J. Biol. Chem. 271 (1996) 5603]. Factors and techniques that influence the use of the Multipin method for successful epitope mapping will be presented.

  10. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele

    2015-01-01

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions. PMID:26216991

  11. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins.

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A; Vendruscolo, Michele

    2015-08-11

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Donnarumma, Danilo; Bartolini, Erika; Borgogni, Erica; Bruttini, Marco; Santini, Laura; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Patanè, Francesco; Biondo, Carmelo; Norais, Nathalie; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    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. PMID:27508302

  14. Identification of immunodominant regions and linear B cell epitopes of the gE envelope protein of varicella-zoster virus.

    Science.gov (United States)

    Fowler, W J; Garcia-Valcarcel, M; Hill-Perkins, M S; Murphy, G; Harper, D R; Jeffries, D J; Burns, N R; Adams, S E; Kingsman, A J; Layton, G T

    1995-12-20

    The envelope proteins of varicella-zoster virus (VZV) are highly immunogenic and one of the most abundant is glycoprotein E (gE). However, its immunodominant regions and epitopes have not been identified. In this study, using human sera from individuals with recent varicella or zoster infections, we have localized antigenic sequences of gE using recombinant hybrid Ty-virus-like particles (VLPs) carrying overlapping fragments of the gE protein. gE(1-134)-VLPs (particles carrying amino acids 1-134 of gE) and, to a lesser extent, gE(101-161)-VLPs were found to be the most antigenic when tested by Western blotting and ELISA. Other fragments of gE (spanning residues 161-623) showed weak or no antigenicity. Pepscan analysis of human sera on overlapping synthetic peptides representing residues 1-135 of gE revealed that the most antigenic region was between residues 50 and 135. Three immunodominant sequences (residues 86-105, 116-135, and, to a lesser extent, 56-75) were detected using sera from both varicella and zoster patients. All sera from varicella, but not zoster, patients reacted strongly with an epitope in peptide 66-85. Other epitopes were recognized weakly by some varicella or zoster sera. More sera need to be tested to assess the potential disease specificity of these epitopes. The neutralizing monoclonal antibody (MAb) IF-B9 reacted with residues 71-90; however, another neutralizing MAb, SG1A, which bound to both gE(1-134)-VLPs and gE(101-161)-VLPs did not bind to any peptide. The identification of immunodominant sequences of gE will help toward the development of a subunit VZV vaccine. PMID:8553555

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

    Science.gov (United States)

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

    2016-08-01

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

  16. Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres

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    Murata Yoshihiko

    2009-06-01

    Full Text Available Abstract Background Human papillomavirus (HPV L1 capsid proteins can self-assemble into pentamers (capsomeres that are immunogenic and can elicit neutralizing antibodies. Structural modelling of L1 inter-pentameric interactions predicts that helix 4 (h4 of each of the five L1 monomers project laterally and outwards from the pentamer. We sought to utilize HPV L1 capsomeres as a vaccine platform by engineering heterologous epitopes within L1 derivatives deleted for h4 domain. Results We used baculovirus – infected Trichoplusia ni cells and ultracentrifugation to synthesize and purify three 16L1 derivatives: one bearing a short deletion (amino acids 404–436 encompassing the h4 domain, and two others, each bearing a conserved neutralizing epitope of the human respiratory syncytial virus (RSV fusion (F protein (residues 255–278 and 423–436 that was substituted for the deleted L1 h4 domain residues. Each of the three capsomere derivatives was recognized by anti-L1 antibodies, while two bearing the RSV F-derived moieties were recognized by anti-RSV F antibodies. All three L1 derivatives formed ring-like structures that were similar in morphology and size to those described for native 16L1 capsomeres. When injected into mice, each of the capsomere derivatives was immunogenic with respect to L1 protein, and immunization with chimeric L1-RSV F pentamers resulted in RSV non-neutralizing antisera that recognized purified RSV F protein in immunoblots. Conclusion HPV L1 monomers bearing heterologous epitopes within the L1 h4 region can self-assemble into capsomeres that elicit antibody response against such non-HPV encoded epitopes. Thus, the L1 h4 region can function as a novel antigen display site within the L1 pentamer, which in turn may serve as a potential vaccine template.

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

    Science.gov (United States)

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Donnarumma, Danilo; Bartolini, Erika; Borgogni, Erica; Bruttini, Marco; Santini, Laura; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Patanè, Francesco; Biondo, Carmelo; Norais, Nathalie; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    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. PMID:27508302

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

    Directory of Open Access Journals (Sweden)

    Stephanie eAscough

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  20. T-cell epitope finding on EPHA2 for further glioma vaccine development: An immunomics study

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2011-01-01

    Full Text Available Background: Glioma is a deadly neurological tumor. For modern management of glioma, glioma vaccinotherapy is the new concept. Materials and Methods: Based on present biomedical technique, the identification of T-cell epitopes via MHC mapping can help clarify the inter-relationship of tumor and immune system. This process can be performed using advanced immunoinformatics technique. Results: Here, the author performs an immunoinformatics analysis to find alternative epitopes for glioma-related antigen, EPHA2. Conclusion: After complete manipulation on EPHA2 molecules, the five best epitopes were derived.

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

    Science.gov (United States)

    Hou, Minbo; Zhou, Defang; Li, Gen; Guo, Huijun; Liu, Jianzhu; Wang, Guihua; Zheng, Qiankun; Cheng, Ziqiang

    2016-03-01

    Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry.

  2. Different Culture Media Affect Proliferation, Surface Epitope Expression, and Differentiation of Ovine MSC.

    Science.gov (United States)

    Adamzyk, Carina; Emonds, Tanja; Falkenstein, Julia; Tolba, René; Jahnen-Dechent, Wilhelm; Lethaus, Bernd; Neuss, Sabine

    2013-01-01

    Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC). Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS) content and in the addition of supplements and/or additional epidermal growth factor (EGF). We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid) determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i) considerable donor to donor variations, (ii) protocol-dependent variations, and (iii) variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation.

  3. Schistosoma japonicum:Isolation and Identification of Peptides Mimicking Ferritin Epitopes from Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    Lian-Fei TANG; Xin-Yuan YI; Xian-Fang ZENG; Lin-Qian WANG; Shun-Ke ZHANG

    2004-01-01

    In an attempt to isolate and identify the antigenic epitopes on ferritin of Schistosoma japonicum(SjFer)and to test their protective potentiality against Schistosomajaponicum(S.j),polyclonal antisera against SjFer was prepared to screen a 12-mer random peptide library.Three rounds of biopanning were performed and resulted in an enrichment.Six peptides selected randomly from the third elute were all found to be positive by evaluating the binding to anti-SjFer sera by ELISA and Western blotting.Three amino acid sequences were deduced from the six phage clones by sequencing.When they were used to vaccinate mice,the three peptides could induce significant reduction in adult worms(26.7% ,20.4%,and 25.9%)as well as in liver eggs per gram(LEPG)(40.0%,38.2%,and 40.8%).This result showed that three mimotopes on SjFer were obtained and they could induce significant protective efficacy against S.j.

  4. Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology

    Institute of Scientific and Technical Information of China (English)

    Ying Gu; Jun Zhang; Ying-Bing Wang; Shao-Wei Li; Hai-Jie Yang; Wen-Xin Luo; Ning-Shao Xia

    2004-01-01

    AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3.METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E. coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor.RESULTS: Twenty-one positive monoclonal phages (10for 8CL1, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N′-His-Pro-Thr-LeuLeu-Arg-Ile-C′, named 8C11A) and 8H3 (N′-Ser-Ile-LeuPro- Tyr-Pro-Tyr-C′, named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E. coli.The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemosynthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor.CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short

  5. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

    Institute of Scientific and Technical Information of China (English)

    Bingxin; Zhao; Xiaoxia; Pan; Yumei; Teng; Wenyue; Xia; Jing; Wang; Yuling; Wen; Yuanding; Chen

    2015-01-01

    VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  6. An overview of bioinformatics tools for epitope prediction: implications on vaccine development.

    Science.gov (United States)

    Soria-Guerra, Ruth E; Nieto-Gomez, Ricardo; Govea-Alonso, Dania O; Rosales-Mendoza, Sergio

    2015-02-01

    Exploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned.

  7. The epitopes in wheat proteins for defining toxic units relevant to human health.

    Science.gov (United States)

    Juhász, Angéla; Gell, Gyöngyvér; Békés, Frank; Balázs, Ervin

    2012-11-01

    Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

  8. Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

    Science.gov (United States)

    Yamniuk, Aaron P; Suri, Anish; Krystek, Stanley R; Tamura, James; Ramamurthy, Vidhyashankar; Kuhn, Robert; Carroll, Karen; Fleener, Catherine; Ryseck, Rolf; Cheng, Lin; An, Yongmi; Drew, Philip; Grant, Steven; Suchard, Suzanne J; Nadler, Steven G; Bryson, James W; Sheriff, Steven

    2016-07-17

    Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications. PMID:27216500

  9. 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...... 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...... 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....

  10. Two novel neutralizing antigenic epitopes of the s1 subunit protein of a QX-like avian infectious bronchitis virus strain Sczy3 as revealed using a phage display peptide library.

    Science.gov (United States)

    Zou, Nianli; Xia, Jing; Wang, Fuyan; Duan, Zhenzhen; Miao, Dan; Yan, Qigui; Cao, Sanjie; Wen, Xintian; Liu, Ping; Huang, Yong

    2015-11-15

    The spike (S) protein of the infectious bronchitis virus (IBV) plays a central role in the pathogenicity, the immune antibody production, serotype and the tissue tropism. In this study, we generate 11 monoclonal antibodies (mAbs) against S1 subunit of IBV Sczy3 strain, and two mAbs 1D5 and 6A12 were positive in indirect ELISA against both His-S1 protein and the purified whole viral antigen. MAb 6A12 and 1D5 could recognized by other 10 IBV strains (IBVs) from five different genotypes, except that 1D5 had a relatively low reaction with two of the 10 tested IBVs. End-point neutralizing assay performed in chicken embro kidney (CEK) cells revealed that the neutralization titer of 6A12 and 1D5 against Sczy3 reached 1:44.7 and 1:40.6, respectively. After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 1D5 and 6A12, which corresponded to the amino acid sequences (87)PPQGMAW(93) and (412)IQTRTEP(418), respectively, in the IBV S1 subunit. Sequences comparison revealed that epitope (412)IQTRTEP(418) was conserved among IBVs, while the epitope (87)PPQGMAW(93) was relatively variable among IBVs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections.

  11. 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.

  12. Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

    Science.gov (United States)

    Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

    2016-06-01

    Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants. PMID:26922739

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Newman Mark J

    2006-03-01

    Full Text Available Abstract Background T cells recognize a complex between a specific major histocompatibility complex (MHC molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. Results To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population. Conclusion We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine, and also minimizing the variability of coverage obtained or projected in different ethnic groups.

  15. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens

    OpenAIRE

    Caro-Aguilar, Ivette; Rodríguez, Alexandra; Calvo-Calle, J. Mauricio; Guzmán, Fanny; De La Vega, Patricia; Elkin Patarroyo, Manuel; Galinski, Mary R.; Moreno, Alberto

    2002-01-01

    Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. viva...

  16. Conformational epitopes of myelin oligodendrocyte glycoprotein are targets of potentially pathogenic antibody responses in multiple sclerosis

    OpenAIRE

    Menge Til; Lalive Patrice H; von Büdingen H -Christian; Genain Claude P

    2011-01-01

    Abstract Background Myelin/oligodendrocyte glycoprotein (MOG) is a putative autoantigen in multiple sclerosis (MS). Establishing the pathological relevance and validity of anti-MOG antibodies as biomarkers has yielded conflicting reports mainly due to different MOG isoforms used in different studies. Because epitope specificity may be a key factor determining anti-MOG reactivity we aimed at identifying a priori immunodominant MOG epitopes by monoclonal antibodies (mAbs) and at assessing clini...

  17. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    Science.gov (United States)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  18. 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;

    between high affinity and high stability peptides bound by the highly expressed SLA molecules, SLA-1*0401, SLA-2*0401, and SLA-3*0401, using a luminescence oxygen channeling (LOCI) and a scintillation proximity assay, respectively. With this procedure, high affinity and highly stable SLA peptide epitopes...... can be identified within a given viral genome, along with the elimination of hundreds, or even thousands, of peptide sequences, which are not likely to be bound. Applying these methods can save enormous amounts of time and costs of epitope discovery studies and MHC binding analysis not only in swine...

  19. Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses

    Science.gov (United States)

    Cioni, M.; Leboeuf, C.; Comoli, P.; Ginevri, F.

    2016-01-01

    Uncontrolled BK polyomavirus (BKPyV) replication in kidney transplant recipients (KTRs) causes polyomavirus‐associated nephropathy and allograft loss. Reducing immunosuppression is associated with clearing viremia and nephropathy and increasing BKPyV‐specific T cell responses in most patients; however, current immunoassays have limited sensitivity, target mostly CD4+ T cells, and largely fail to predict onset and clearance of BKPyV replication. To characterize BKPyV‐specific CD8+ T cells, bioinformatics were used to predict 9mer epitopes in the early viral gene region (EVGR) presented by 14 common HLAs in Europe and North America. Thirty‐nine EVGR epitopes were experimentally confirmed by interferon‐γ enzyme‐linked immunospot assays in at least 30% of BKPyV IgG–seropositive healthy participants. Most 9mers clustered in domains, and some were presented by more than one HLA class I, as typically seen for immunodominant epitopes. Specific T cell binding using MHC class I streptamers was demonstrated for 21 of 39 (54%) epitopes. In a prospective cohort of 118 pediatric KTRs, 19 patients protected or recovering from BKPyV viremia were experimentally tested, and 13 epitopes were validated. Single HLA mismatches were not associated with viremia, suggesting that failing immune control likely involves multiple factors including maintenance immunosuppression. Combining BKPyV load and T cell assays using immunodominant epitopes may help in evaluating risk and reducing immunosuppression and may lead to safe adoptive T cell transfer. PMID:26663765

  20. B-cell epitope mapping for the design of vaccines and effective diagnostics

    Directory of Open Access Journals (Sweden)

    Tarek A. Ahmad

    2016-01-01

    Full Text Available The increasing resistance of many microbial strains to antibiotics, delayed laboratory results, and side effects of many chemotherapeutics has raised the need to search for sensitive diagnostics and new prophylactic strategies especially prevention by vaccination. Understanding the epitope/antibody interaction is the key to constructing potent vaccines and effective diagnostics. B-cell epitope mapping is a promising approach to identifying the main antigenic determinants of microorganisms, in special concern the discontinuous conformational ones. Epitope-based vaccines have remarkable privilege over the conventional ones since they are specific, able to avoid undesirable immune responses, generate long lasting immunity, and are reasonably cheaper. This up-to-date review discusses and compares the different physical, computational, and molecular methods that have been used in epitope mapping. The role of each method in the identification of potent epitopes in viruses, bacteria, fungi, parasites, as well as human diseases are tagged and documented. Simultaneously, frequent combinatorial methods are highlighted. The article aims to assist researchers to design the most suitable protocol for mapping their B-cell epitopes.

  1. Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

    Institute of Scientific and Technical Information of China (English)

    Shahla; Shahsavandi; Mohammad; Majid; Ebrahimi; Kaveh; Sadeghi; Homayoon; Mahravani

    2015-01-01

    Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.

  2. A Novel Contraceptive Vaccine:Design and Synthesis of the Chimeric Peptide Containing Multivalent Sperm-Specific Epitopes

    Institute of Scientific and Technical Information of China (English)

    何畏; 梁志清; 史常旭; 李玉清

    2001-01-01

    Objective To develop a novel multivalent chimeric peptide vaccine for bisexual fertility regulation Materials & Methods On the basis of the amino acid sequence of the two peptides respectively selected from the mouse sperm/testis-specific proteins SP17 and Cyritestin, and one T cell epitope in bovine ribonuclease (RNase), a novel chimeric peptide consisting of 35 amino acid was designed and subsequently synthesized on the 430A peptide synthesizer. After being emulified with the equivalence Freund's adjuvant, the peptide with 35 amino acid residues was used to immunogenity the female BALB/c mice to investigate its immunity.Results The peptide was successfully synthesized, after being purified in high performance liquid chromatography (HPLC), its purity reached 95%. The specific antisera collected from the immunogenity mice could identify the corresponding proteins of testis tissues of mice, rats and human. The highest specific IgG titer in serum was 1:6 000, while the IgA titer in the washing of vaginal mucous membrane was 1:300.Conclusion The antibodies from the peptide with specific amino acid sequence can identify the original antigens, and stimulate powerful specific humoral immunity in mice. It provides a experimental bases for polyvalent contraceptive vaccine study.

  3. Antigenic topology of the P29 surface lipoprotein of Mycoplasma fermentans: differential display of epitopes results in high-frequency phase variation.

    Science.gov (United States)

    Theiss, P; Karpas, A; Wise, K S

    1996-05-01

    Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important

  4. T-cell epitope vaccine design by immunoinformatics.

    Science.gov (United States)

    Patronov, Atanas; Doytchinova, Irini

    2013-01-08

    Vaccination is generally considered to be the most effective method of preventing infectious diseases. All vaccinations work by presenting a foreign antigen to the immune system in order to evoke an immune response. The active agent of a vaccine may be intact but inactivated ('attenuated') forms of the causative pathogens (bacteria or viruses), or purified components of the pathogen that have been found to be highly immunogenic. The increased understanding of antigen recognition at molecular level has resulted in the development of rationally designed peptide vaccines. The concept of peptide vaccines is based on identification and chemical synthesis of B-cell and T-cell epitopes which are immunodominant and can induce specific immune responses. The accelerating growth of bioinformatics techniques and applications along with the substantial amount of experimental data has given rise to a new field, called immunoinformatics. Immunoinformatics is a branch of bioinformatics dealing with in silico analysis and modelling of immunological data and problems. Different sequence- and structure-based immunoinformatics methods are reviewed in the paper.

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

    Science.gov (United States)

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

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine. PMID:26552001

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

    Science.gov (United States)

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

    2015-12-16

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

  7. A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

    Science.gov (United States)

    Jones, Tim D.; Hearn, Arron R.; Holgate, Robert G.E.; Kozub, Dorota; Fogg, Mark H.; Carr, Francis J.; Baker, Matthew P.; Lacadena, Javier; Gehlsen, Kurt R.

    2016-01-01

    Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics. PMID:27578884

  8. PreS1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac™ vaccine and their relation to the antibody response to hepatitis B surface antigen

    Directory of Open Access Journals (Sweden)

    Madalinski Kazimierz

    2009-01-01

    Full Text Available Abstract Background Sci-B-Vac™ is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing hepatitis B surface antigen (HBsAg as well as preS1 and preS2 antigens. Few studies have been performed on the antibody responses to preS1 in relation to the antibody to hepatitis B surface antigen (anti-HBs response during immunisation of healthy children with preS-containing vaccines. Results In this study 28 healthy newborns were randomly selected to receive either 2.5 ug or 5.0 ug of the Sci-B-Vac vaccine. Children received three doses of vaccine according to a 0-, 1-, 6-month scheme. Antibodies against the S-protein and three synthetic peptides mimicking three B-cell preS1 epitopes, (21–32 amino acid epitope, (32–47 amino acid epitope and the C-terminal (amino acid epitope 94–117 were determined at 6 and 9 months. Fourteen (50% of the 28 newborns had detectable levels of anti-preS1 (21–32 antibodies; 15 (54% were anti-preS1 (32–47 reactive and 12 (43% were anti-preS1 (94–117 reactive at 6 or 9 months after initiation of the vaccination. Significantly higher levels of anti-HBs were observed in the sera of patients with detectable anti-preS1 (32–47 reactivity (24 550 ± 7375 IU/L, mean ± SEM as compared with the non-reactive sera (5991 ± 1530 IU/L, p Conclusion Recognition of several preS1 epitopes, and in particular, the epitope contained within the second half of the hepatocyte binding site localised in the hepatitis B surface protein of the third-generation hepatitis B vaccine is accompanied by a more pronounced antibody response to the S-gene-derived protein in healthy newborns.

  9. Identification of a naturally processed cytotoxic CD8 T-cell epitope of coxsackievirus B4, presented by HLA-A2.1 and located in the PEVKEK region of the P2C nonstructural protein.

    Science.gov (United States)

    Varela-Calvino, Ruben; Skowera, Ania; Arif, Sefina; Peakman, Mark

    2004-12-01

    The adaptive immune system generates CD8 cytotoxic T lymphocytes (CTLs) as a major component of the protective response against viruses. Knowledge regarding the nature of the peptide sequences presented by HLA class I molecules and recognized by CTLs is thus important for understanding host-pathogen interactions. In this study, we focused on identification of a CTL epitope generated from coxsackievirus B4 (CVB4), a member of the enterovirus group responsible for several inflammatory diseases in humans and often implicated in the triggering and/or acceleration of the autoimmune disease type 1 diabetes. We identified a 9-mer peptide epitope that can be generated from the P2C nonstructural protein of CVB4 (P2C(1137-1145)) and from whole virus by antigen-presenting cells and presented by HLA-A2.1. This epitope is recognized by effector memory (gamma interferon [IFN-gamma]-producing) CD8 T cells in the peripheral blood at a frequency of responders that suggests that it is a major focus of the anti-CVB4 response. Short-term CD8 T-cell lines generated against P2C(1137-1145) are cytotoxic against peptide-loaded target cells. Of particular interest, the epitope lies within a region of viral homology with the diabetes-related autoantigen, glutamic acid decarboxylase-65 (GAD(65)). However, P2C(1137-1145)-specific cytotoxic T lymphocyte (CTL) lines were not activated to produce IFN-gamma by the GAD(65) peptide homologue and did not show cytotoxic activity in the presence of appropriately labeled targets. These results describe the first CD8 T-cell epitope of CVB4 that will prove useful in the study of CVB4-associated disease. PMID:15564450

  10. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex : characterization of the epitopes

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Berndt, M C; Dawes, I; Chesterman, C N; Chong, B H

    1998-01-01

    Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to ch

  11. Analyzing the H19- and T65-epitopes in 38 kd phosphorylated protein of Marek's disease viruses and comparing chicken immunological reactions to viruses point-mutated in the epitopes

    Institute of Scientific and Technical Information of China (English)

    CUI Zhizhong; ZHANG Zhi; QIN Aijian; Lee Lucy F

    2004-01-01

    DNA sequencing analysis in 38 kd phosphorylated protein (pp38) ORF of Marek's disease viruses (MDV) indicated that all tested 10 virulent strains with different pathotypes had 'A' at base #320 and glutamine at aa#107 while reacted with monoclonal antibody (Mab) H19 in indirect fluorescence antibody test (IFA). However, vaccine strain CVI988 had 'G' at base#320 and arginine at aa#107 instead, when it was negative in IFA with Mab H19. Some strains were also reactive with Mab T65 in IFA while there was 'G' at base #326 and glycine at aa#109, but the other strains, which had 'A' at base #326 and glutamic acid at aa#109, did not react with Mab T65. By comparison of CVI988 to its point mutants CVI/rpp38(AG) and CVI/rpp38(AA) with 1 or 2 base(s) changes at bases #320 and /or #326 of pp38 gene for their reactivity with Mab H19 and T65, it was confirmed that the glutamine at aa#107 and glycine at aa#109 were critical to epitopes H19 and T65 respectively. Immuno-reactions to MDV were compared in SPF chickens inoculated with cloned CVI988 and its mutant CVI/rpp38(AG). It was found that antibody responses to MDV in chickens inoculated with CVI/rpp38(AG) were delayed and significantly lower than that in chickens inoculated with the native CVI988. By differential comparison of antibody titers to different antigens, a third epitope specific to CVI988 and dependent on arginine at aa#107 was suggested to be responsible for the big difference in antibody responses induced by native CVI988 and its mutant.

  12. 应用生物信息学分析大米过敏原RAG1的B细胞表位(英文)%Bioinformatics Analysis on B cell Epitopes of Rice Allergen RAG1

    Institute of Scientific and Technical Information of China (English)

    李燕芳; 何颖; 邹泽红

    2012-01-01

    [Objective] To predict the secondary structure and B cell epitopes of the rice major allergen RAG1. [Method] The amino acid sequence of rice allergen RAG1 was acquired from Expasy protein database. The secondary structure of RAG1 was predicted by DNAStar Protean software with Gamier-Robson program, Chou-Fasman program and Karplus-Schulz program; the B cell epitopes of RAG1 was predicted with the Kyte Doolittle hydrophilic program, Emini surface accessibility program and Jameson-Wolf antigenic index program. [Result] The predictions on secondary structure and B cell epitopes showed that the regions of 33-44, 119-129, 155-163 were the dominant B cell epitopes. [Conclusion] This study predicted the potential dominant B cell epitopes in rice allergen RAG1 by comprehensive use of multi-methods and multi-parameters, and provided a theoretical basis for further researches on identification, antigen modification and epitope vaccine design of RAG1 B cell epitopes.%[目的]预测大米主要过敏原RAG1的二级结构及B细胞表位。[方法]从Expasy蛋白质数据库中获得编码大米过敏原RAG1的氨基酸序列并以此氨基酸序列为基础,通过DNAStarProtean软件,采用Gamier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测RAG1的二级结构,用Kyte-Doolittle亲水性方案、Emini表面可及性方案、Jameson-Wolf抗原性指数方案综合预测RAG1的B细胞表位。[结果]对大米过敏原RAG1的二级结构和B细胞表位预测的结果表明,第33~44、119~129、155~163区段可能是B细胞的优势表位。[结论]本研究综合应用多方法多参数预测大米过敏原RAG1潜在的B细胞优势表位,为RAG1B细胞表位的进一步鉴定及其抗原性改造、表位疫苗设计等研究提供了理论依据。

  13. Discovery of common marburgvirus protective epitopes in a BALB/c mouse model

    Directory of Open Access Journals (Sweden)

    Olinger Gene G

    2009-08-01

    Full Text Available Abstract Background Marburg virus (MARV causes acute hemorrhagic fever that is often lethal, and no licensed vaccines are available for preventing this deadly viral infection. The immune mechanisms for protection against MARV are poorly understood, but previous studies suggest that both antibodies and T cells are required. In our study, we infected BALB/c mice with plaque-purified, nonlethal MARV and used overlapping peptides to map H2d-restricted CD8+ T-cell epitopes. Methods Splenocytes from mice infected with nonlethal MARV were harvested and stimulated with multiple overlapping 15-mer peptide pools, and reactive CD8+ T cells were evaluated for antigen specificity by measuring upregulation of CD44 and interferon-γ expression. After confirming positive reactivity to specific 15-mer peptides, we used extrapolated 9-mer epitopes to evaluate the induction of cytotoxic T-cell responses and protection from lethal MARV challenge in BALB/c mice. Results We discovered a CD8+ T-cell epitope within both the MARV glycoprotein (GP and nucleoprotein (NP that triggered cytotoxic T-cell responses. These responses were also protective when epitope-specific splenocytes were transferred into naïve animals. Conclusion Epitope mapping of MARV GP, NP, and VP40 provides the first evidence that specific MARV-epitope induction of cellular immune responses is sufficient to combat infection. Establishment of CD8+ T-cell epitopes that are reactive to MARV proteins provides an important research tool for dissecting the significance of cellular immune responses in BALB/c mice infected with MARV.

  14. Epitope characterization of an anti-PD-L1 antibody using orthogonal approaches.

    Science.gov (United States)

    Hao, Gang; Wesolowski, John S; Jiang, Xuliang; Lauder, Scott; Sood, Vanita D

    2015-04-01

    The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques. PMID:25664688

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

    OpenAIRE

    2014-01-01

    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 ab...

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion......, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1¿4)-ß- -galactan epitope occurring...

  17. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

    Institute of Scientific and Technical Information of China (English)

    TANG; Yuyang

    2001-01-01

    [1]Hill, A. V., Elvin, J., Willis, A. C. et al., Molecular analysis of the association of HLA-B53 and resistance to severe malaria, Nature, 1992, 360: 434.[2]Perlmann, P., Miller, L., Fogarty/WHO international conference on cellular mechanisms in malaria immunity, Immun. Letter, 1990, 25: 1.[3]Perkus, M. E., Tartaglia, J., Paoletti, E. et al., Poxvirus-based vaccine candidates for cancer, AIDS and other infectious diseases, J. Leukocyte Biol., 1995, 58(1): 1.[4]Shen, H., Slifka, M. K., Matloubian, M. et al., Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity, Proc. Natl. Acad. Sci. USA, 1995, 92: 3987.[5]Waine, G. J., McManus, D. P., Nucleic acids: vaccines of the future, Parasitol Today, 1995, 11: 113.[6]Whitton, J. L., Sheng, N., Oldstone, M. B. A. et al., A "string-of beads" vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge, J. Virol., 1993, 67: 348.[7]Lalvani, A., Aidoo, M., Allsopp, C. E. et al., An-HLA-based approach to design of a CTL-inducing vaccine against Plasmodium falciparum, Res. Immunol., 1994, 145: 461.[8]Sidney, J., Grey, H. M., Kubo, R. T. et al., Practical, biochemical and evolutionary implications of the discovery of HLA class I supermotifs, Immunology Today, 1996, 17: 261.[9]Thomson, S. A., Elliott, S. L., Sherritt, M. A. et al., Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes, J. Immun., 1996, 157: 822.[10] Hanke, T., Schneider, J., Gilbert, S. C. et al., DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice, Vaccine, 1998, 16: 426.[11] Townsend, A. R. M., Rothbard, J., Gotch, F. M. et al., The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides, Cell, 1986, 44: 959.[12] Ojcius, D. M., Abastado, J. P., Casrouge, A. et al., Dissociation of

  18. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

    Directory of Open Access Journals (Sweden)

    Kuhn Andreas

    2011-09-01

    Full Text Available Abstract Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

  19. Fucose-binding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes.

    Science.gov (United States)

    Audfray, Aymeric; Claudinon, Julie; Abounit, Saïda; Ruvoën-Clouet, Nathalie; Larson, Göran; Smith, David F; Wimmerová, Michaela; Le Pendu, Jacques; Römer, Winfried; Varrot, Annabelle; Imberty, Anne

    2012-02-01

    Burkholderia ambifaria is generally associated with the rhizosphere of plants where it has biocontrol effects on other microorganisms. It is also a member of the Burkholderia cepacia complex, a group of closely related bacteria that cause lung infections in immunocompromised patients as well as in patients with granulomatous disease or cystic fibrosis. Our previous work indicated that fucose on human epithelia is a frequent target for lectins and adhesins of lung pathogens (Sulák, O., Cioci, G., Lameignère, E., Balloy, V., Round, A., Gutsche, I., Malinovská, L., Chignard, M., Kosma, P., Aubert, D. F., Marolda, C. L., Valvano, M. A., Wimmerová, M., and Imberty, A. (2011) PLoS Pathog. 7, e1002238). Analysis of the B. ambifaria genome identified BambL as a putative fucose-binding lectin. The 87-amino acid protein was produced recombinantly and demonstrated to bind to fucosylated oligosaccharides with a preference for αFuc1-2Gal epitopes. Crystal structures revealed that it associates as a trimer with two fucose-binding sites per monomer. The overall fold is a six-bladed β-propeller formed by oligomerization as in the Ralstonia solanacearum lectin and not by sequential domains like the fungal fucose lectin from Aleuria aurantia. The affinity of BambL for small fucosylated glycans is very high as demonstrated by microcalorimetry (K(D) < 1 μM). Plant cell wall oligosaccharides and human histo-blood group oligosaccharides H-type 2 and Lewis Y are bound with equivalent efficiency. Binding to artificial glycosphingolipid-containing vesicles, human saliva, and lung tissues confirmed that BambL could recognize a wide spectrum of fucosylated epitopes, albeit with a lower affinity for biological material from nonsecretor individuals.

  20. Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

    Science.gov (United States)

    Fallahi, Firouzeh; Wandeler, Alexander I; Nadin-Davis, Susan A

    2016-07-15

    The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus. PMID:27132040

  1. Analysis of structures and epitopes of a novel secreted protein MYR1 in Toxoplasma gondii.

    Science.gov (United States)

    Zhou, Jian; Lu, Gang; He, Shenyi

    2016-01-01

    Toxoplasma gondii (Nicolle et Manceaux, 1908) is an obligate intracellular apicomplexan parasite and can infect warmblooded animals and humans all over the world. Development of effective vaccines is considered the only ideal way to control infection with T. gondii. However, only one live vaccine is commercially available for use in sheep and goats. Thus more effective antigenic proteins are searched for. In the present study we report a novel protein by secreted T. gondii termed Myc regulation 1 (MYR1). The physical and chemical characteristics, epitopes, hydrophilicity and functional sites of MYR1 were analysed by multiple bioinformatic approaches. The 3D models of MYR1 proteins were constructed and analysed. Furthermore, liner B-cell epitopes and T-cell epitopes of MYR1 protein and SAG1 were predicted. Compared to SAG1, MYR1 with good B-cell epitopes and T-cell epitopes had a potentiality to become a more successful vaccine against T. gondii. The bioinformatics analysis of MYR1 proteins could laid the foundation for further studies of its biological function experimentally and provide valuable information necessary for a better prevention and treatment of toxoplasmosis. PMID:27580381

  2. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans.

    Science.gov (United States)

    Chou, Meng-Yun; Fogelstrand, Linda; Hartvigsen, Karsten; Hansen, Lotte F; Woelkers, Douglas; Shaw, Peter X; Choi, Jeomil; Perkmann, Thomas; Bäckhed, Fredrik; Miller, Yury I; Hörkkö, Sohvi; Corr, Maripat; Witztum, Joseph L; Binder, Christoph J

    2009-05-01

    Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

  3. Anti-beta-2 glycoprotein I epitope specificity: from experimental models to diagnostic tools.

    Science.gov (United States)

    Meroni, P L

    2016-07-01

    Beta-2 glycoprotein I (β2GPI) is the main antigenic target for anti-phospholipid antibodies (aPL), the serological markers of anti-phospholipid syndrome (APS). Conformational changes of the molecule seem to be essential for exposing the cryptic epitope for aPL binding and to trigger pathogenic pathways. There is increasing evidence that a conformational epitope located in the Domain I (DI) of the molecule is the main epitope targeted by human autoantibodies. The pathogenic role of the DI epitope has been recently supported by in vivo models and by immuno-histopathological findings in APS patients. Antibodies targeting β2GPI-DI are more frequently detected in patients with full-blown APS compared to asymptomatic aPL carriers or patients with infectious diseases who have antibodies directed against the whole molecule. Anti-DI antibodies are positively correlated with medium to high titres of aPL, with the presence of lupus anticoagulant and thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Although preliminary results suggest that anti-DI antibodies can be detected by different assays in a comparable manner, further prospective studies are needed to support their use in the clinical setting and their predictive value. PMID:27252268

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

    Directory of Open Access Journals (Sweden)

    Bifang He

    2013-01-01

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

  5. Identification of immunogenic HLA-B7 "Achilles' heel" epitopes within highly conserved regions of HIV

    DEFF Research Database (Denmark)

    De Groot, Anne S; Rivera, Daniel S; McMurry, Julie A;

    2008-01-01

    previously described as restricted by B7. The HLA-B7 restricted epitopes discovered using this in silico screening approach are highly conserved across strains and clades of HIV as well as conserved in the HIV genome over the 20 years since HIV-1 isolates were first sequenced. This study demonstrates......Genetic polymorphisms in class I human leukocyte antigen molecules (HLA) have been shown to determine susceptibility to HIV infection as well as the rate of progression to AIDS. In particular, the HLA-B7 supertype has been shown to be associated with high viral loads and rapid progression...... to disease. Using a multiplatform in silico/in vitro approach, we have prospectively identified 45 highly conserved, putative HLA-B7 restricted HIV CTL epitopes and evaluated them in HLA binding and ELISpot assays. All 45 epitopes (100%) bound to HLA-B7 in cell-based HLA binding assays: 28 (62%) bound...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington......, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme...... 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....

  7. 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......Survivin is overexpressed in several malignancies and in tumor-associated endothelium making it an attractive target for therapeutic cytotoxic T-cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence...... in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin-directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model....

  8. Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine

    DEFF Research Database (Denmark)

    De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T;

    2012-01-01

    Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response...... and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context...... of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs...

  9. The Utilization and Limitation of CD133 Epitopes in Lung Cancer Stem Cells Research

    Directory of Open Access Journals (Sweden)

    Yin CHEN

    2011-10-01

    Full Text Available Lung cancer is one of the most common tumor, which lacks of effective clinical treatment to lead to desirable prognosis. According to cancer stem cell hypothesis, lung cancer stem cells are considered to be responsible for carcinogenesis, development, metastasis, recurrence, invasion, resistance to chemotherapy and radiotherapy of lung cancer. In recent years, more and more institutes used glycosylated CD133 epitopes to define, isolate, purify lung cancer stem cells. However, along with deeply research, the application of CD133 epitopes in lung cancer stem cell research is questioned. The utilization and limitation of CD133 epitopes in lung cancer stem cells research for the past few years is summaried in this review.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Mature Epitope Density (MED). Our method, though simple, is capable of identifying promising vaccine targets. Our online software implementation provides a computationally light and reliable analysis of bacterial exoproteins and their potential for vaccines or diagnosis projects against pathogenic......BACKGROUND: Current immunological bioinformatic approaches focus on the prediction of allele-specific epitopes capable of triggering immunogenic activity. The prediction of major histocompatibility complex (MHC) class I epitopes is well studied, and various software solutions exist for this purpose...... organisms. We evaluated our computational approach by using the Mycobacterium tuberculosis (Mtb) H37Rv exoproteome as a gold standard model. A literature search was carried out on 60 out of 553 Mtb's predicted exoproteins, looking for previous experimental evidence concerning their possible antigenicity...

  11. The epitope of monoclonal antibodies blocking erythrocyte invasion by Plasmodium falciparum map to the dimerization and receptor glycan binding sites of EBA-175.

    Directory of Open Access Journals (Sweden)

    Xavier Ambroggio

    Full Text Available The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 β-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 β-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 β-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.

  12. Induction of epitope-specific neutralizing antibodies against West Nile virus.

    Science.gov (United States)

    Oliphant, Theodore; Nybakken, Grant E; Austin, S Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H; Pierson, Theodore C; Diamond, Michael S

    2007-11-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.

  13. Induction of Epitope-Specific Neutralizing Antibodies against West Nile Virus▿

    Science.gov (United States)

    Oliphant, Theodore; Nybakken, Grant E.; Austin, S. Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H.; Pierson, Theodore C.; Diamond, Michael S.

    2007-01-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies. PMID:17715236

  14. Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy.

    Science.gov (United States)

    Fresquet, Maryline; Jowitt, Thomas A; Gummadova, Jennet; Collins, Richard; O'Cualain, Ronan; McKenzie, Edward A; Lennon, Rachel; Brenchley, Paul E

    2015-02-01

    Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.

  15. Homology Modeling and Conformational Epitope Prediction of Envelope Protein of Alkhumra Haemorrhagic Fever Virus

    Directory of Open Access Journals (Sweden)

    Naghmeh Poorinmohammad

    2015-10-01

    Full Text Available Background: The aim of this study was to generate in silico 3D-structure of the envelope protein of AHFV using homology modeling method to further predict its conformational epitopes and help other studies to investigate itsstructural features using the model.Methods: A 3D-structure prediction was developed for the envelope protein of Alkhumra haemorrhagic fever virus (AHFV, an emerging tick-borne flavivirus, based on a homology modeling method using M4T and Modwebservers, as the 3D-structure of the protein is not available yet. Modeled proteins were validated using Modfold 4 server and their accuracies were calculated based on their RSMDs. Having the 3D predicted model with high quality, conformational epitopes were predicted using DiscoTope 2.0.Results: Model generated by M4T was more acceptable than the Modweb-generated model. The global score and Pvalue calculated by Modfold 4 ensured that a certifiable model was generated by M4T, since its global score was almost near 1 which is the score for a high resolution X-ray crystallography structure. Furthermore, itsthe P-value was much lower than 0.001 which means that the model is completely acceptable. Having 0.46 Å rmsd, this model was shown to be highly accurate. Results from DiscoTope 2.0 showed 26 residues as epitopes, forming conformational epitopes of the modeled protein.Conclusion: The predicted model and epitopes for envelope protein of AHFV can be used in several therapeutic and diagnostic approaches including peptide vaccine development, structure based drug design or diagnostic kit development in order to facilitate the time consuming experimental epitope mapping process.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    NARCIS (Netherlands)

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

    2016-01-01

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

  18. 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....... In contrast, the benchmark study by Khan et al. (2008) resulted in 165 conserved 9-mer peptides. Many of the conserved blocks are located consecutively in the proteins. Connecting these blocks resulted in 78 conserved regions. Of the 1551 blocks of 9-mer peptides 110 comprised predicted HLA binder sets...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, "The Immune Epitope Database and Analysis Resource." We describe how we plan to capture, structure......Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance...

  20. P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections.

    Directory of Open Access Journals (Sweden)

    David C Whitacre

    Full Text Available In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs. Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10(6 and provided 80-100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78, which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.

  1. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    OpenAIRE

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.; Landry, Samuel J.

    2014-01-01

    Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-te...

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

    Science.gov (United States)

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

    2015-01-30

    Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays. PMID:25465659

  3. New insights into wheat toxicity: Breeding did not seem to contribute to a prevalence of potential celiac disease's immunostimulatory epitopes.

    Science.gov (United States)

    Ribeiro, Miguel; Rodriguez-Quijano, Marta; Nunes, Fernando M; Carrillo, Jose Maria; Branlard, Gérard; Igrejas, Gilberto

    2016-12-15

    Gluten proteins, namely gliadins, are the primary trigger of the abnormal immune response in celiac disease. It has been hypothesised that modern wheat breeding practices may have contributed to the increase in celiac disease prevalence during the latter half of the 20th century. Our results do not support this hypothesis as Triticum aestivum spp. vulgare landraces, which were not subjected to breeding practices, presented higher amounts of potential celiac disease's immunostimulatory epitopes when compared to modern varieties. Furthermore, high variation between wheat varieties concerning the toxic epitopes amount was observed. We carried out quantitative analysis of gliadin types by RP-HPLC to verify its correlation with the amount of toxic epitopes: ω-type gliadins content explain about 40% of the variation of toxic epitopes in tetraploid wheat varieties. This research provides new insights regarding wheat toxicity and into the controversial idea that human practices may have conducted to an increased exposure to toxic epitopes. PMID:27451149

  4. 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

    was determined using luminescence oxygen channeling (LOCI) and scintillation proximity assay (SPA) for peptides bound by the commonly occurring SLA molecules, SLA-1*0401, SLA-2*0401, and SLA-3*0401. Using these tools, peptides bound by SLA with high affinity and stability were identified from a library of 10.......000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods...... originally developed for analysis of human leukocyte antigen (HLA) presentation of peptides. The methods presented provide a timely and cost-effective approach to CTL epitope discovery that can be applied to diseases of swine and of other mammalian species of interest....

  5. The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines

    DEFF Research Database (Denmark)

    Sørensen, Rikke Baek; Junker, Niels; Kirkin, Alexei;

    2009-01-01

    of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy....

  6. Significance of monoclonal antibodies against the conserved epitopes within non-structural protein 3 helicase of hepatitis C virus.

    Directory of Open Access Journals (Sweden)

    Yixin Bian

    Full Text Available Nonstructural protein 3 (NS3 of hepatitis C virus (HCV, codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192-1459. Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope (1231PTGSGKSTK(1239 (EP05 or core motif (1373IPFYGKAI(1380 (EP21, respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59-79% chronic and weakly with 30-58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.

  7. EpViX: A cloud-based tool for epitope reactivity analysis and epitope virtual crossmatching to identify low immunologic risk donors for sensitized recipients.

    Science.gov (United States)

    Anunciação, Fernando Antonio Costa; Sousa, Luiz Claudio Demes da Mata; da Silva, Adalberto Socorro; Marroquim, Mário Sérgio Coelho; Coelho, Antônio Gilberto Borges; Willcox, Glauco Henrique; de Andrade, João Marcelo Medeiros; Corrêa, Bruno de Melo; Guimarães, Elisabeth Lima; do Monte, Semiramis Jamil Hadad

    2015-11-01

    One of the challenges facing solid organ transplantation programs globally is the identification of low immunological risk donors for sensitized recipients by HLA allele genotype. Because recognition of donor HLA alleles by host antibodies is at the core of organ rejection, the objective of this work was to develop a new version of the EpHLA software, named EpViX, which uses an HLAMatchmaker algorithm and performs automated epitope virtual crossmatching at the initiation of the organ donation process. EpViX is a free, web-based application developed for use over the internet on a tablet, smartphone or computer. This program was developed using the Ruby programming language and the Ruby-on-Rails framework. To improve the user experience, the EpViX software interface was developed based on the best human–computer interface practices. To simplify epitope analysis and virtual crossmatching, the program was integrated with important available web-based resources, such as OPTN, IMGT/HLA and the International HLA Epitope Registry. We successfully developed a program that allows people to work collaboratively and effectively during the donation process by accurately predicting negative crossmatches, saving time and other resources.

  8. EpViX: A cloud-based tool for epitope reactivity analysis and epitope virtual crossmatching to identify low immunologic risk donors for sensitized recipients.

    Science.gov (United States)

    Anunciação, Fernando Antonio Costa; Sousa, Luiz Claudio Demes da Mata; da Silva, Adalberto Socorro; Marroquim, Mário Sérgio Coelho; Coelho, Antônio Gilberto Borges; Willcox, Glauco Henrique; de Andrade, João Marcelo Medeiros; Corrêa, Bruno de Melo; Guimarães, Elisabeth Lima; do Monte, Semiramis Jamil Hadad

    2015-11-01

    One of the challenges facing solid organ transplantation programs globally is the identification of low immunological risk donors for sensitized recipients by HLA allele genotype. Because recognition of donor HLA alleles by host antibodies is at the core of organ rejection, the objective of this work was to develop a new version of the EpHLA software, named EpViX, which uses an HLAMatchmaker algorithm and performs automated epitope virtual crossmatching at the initiation of the organ donation process. EpViX is a free, web-based application developed for use over the internet on a tablet, smartphone or computer. This program was developed using the Ruby programming language and the Ruby-on-Rails framework. To improve the user experience, the EpViX software interface was developed based on the best human–computer interface practices. To simplify epitope analysis and virtual crossmatching, the program was integrated with important available web-based resources, such as OPTN, IMGT/HLA and the International HLA Epitope Registry. We successfully developed a program that allows people to work collaboratively and effectively during the donation process by accurately predicting negative crossmatches, saving time and other resources. PMID:26531328

  9. Bioinformatics Analysis on B Cell Epitopes of Rice Allergen RAG1%应用生物信息学分析大米过敏原RAG1的B细胞表位

    Institute of Scientific and Technical Information of China (English)

    李燕芳; 何颖; 邹泽红

    2012-01-01

    [目的]预测大米主要过敏原RAG1的二级结构及B细胞表位.[方法]从Expasy蛋白质数据库中获得编码大米过敏原RAG1的氨基酸序列并以此氨基酸序列为基础,通过DNAStar Protean软件,采用Gamier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测RAG1的二级结构,用Kyte-Doolittle亲水性方案、Emini表面可及性方案、Jameson-Wolf抗原性指数方案综合预测RAG1的B细胞表位.[结果]对大米过敏原RAG1的二级结构和B细胞表住预测的结果表明,第33 ~44、119 ~129、155~163区段可能是B细胞的优势表位.[结论]该研究综合应用多方法多参数预测大米过敏原RAG1潜在的B细胞优势表位,为RAG1 B细胞表位的进一步鉴定及其抗原性改造、表位疫苗设计等研究提供了理论依据.%[Objective] To predict secondary structure and B cell epilopes of the rice major allergen RAG1. [ Method ] The amino acid sequence of rice allergen RAG1 was acquired from Expasy protein database. The secondary structure of RAG1 was predicted by DNAStar Protean software with Gamier-Robson program, Chou-Fasman program and Karplus-Schulz program; the B cell epitopes of RAG1 was predicted with the Kyte-Doolittle hydrophilic program, Emini surface accessibility program and Jameson-Wolf antigenic index program. [ Result] The predictions on secondary structure and B cell epitopes showed that the regions of 33 -44, 119 - 129, 155 - 163 were the dominant B cell epitopes. [Conclusion] This study predicted the potential dominant B cell epitopes in rice allergen RAG1 by comprehensive use of multi-methods and multi-parameters, and provided a theoretical basis for further researches on identification, antigen modification and epitope vaccine design of RAG1 B cell epitopes.

  10. Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

    Science.gov (United States)

    Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

    2014-10-01

    One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

  11. Human HLA class I- and HLA class II-restricted cloned cytotoxic T lymphocytes identify a cluster of epitopes on the measles virus fusion protein.

    Science.gov (United States)

    van Binnendijk, R S; Versteeg-van Oosten, J P; Poelen, M C; Brugghe, H F; Hoogerhout, P; Osterhaus, A D; Uytdehaag, F G

    1993-01-01

    The transmembrane fusion (F) glycoprotein of measles virus is an important target antigen of human HLA class I- and class II-restricted cytotoxic T lymphocytes (CTL). Genetically engineered F proteins and nested sets of synthetic peptides spanning the F protein were used to determine sequences of F recognized by a number of F-specific CTL clones. Combined N- and C-terminal deletions of the respective peptides revealed that human HLA class I and HLA class II-restricted CTL efficiently recognize nonapeptides or decapeptides representing epitopes of F. Three distinct sequences recognized by three different HLA class II (DQw1, DR2, and DR4/w53)-restricted CTL clones appear to cluster between amino acids 379 and 466 of F, thus defining an important T-cell epitope area of F. Within this same region, a nonamer peptide of F was found to be recognized by an HLA-B27-restricted CTL clone, as expected on the basis of the structural homology between this peptide and other known HLA-B27 binding peptides. PMID:7680390

  12. Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

    Science.gov (United States)

    Mendes, T M; Oliveira, D; Figueiredo, L F M; Machado-de-Avila, R A; Duarte, C G; Dias-Lopes, C; Guimarães, G; Felicori, L; Minozzo, J C; Chávez-Olortegui, C

    2013-06-01

    A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

  13. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants

    Indian Academy of Sciences (India)

    Noemi Cerovska; Hana Hoffmeisterova; Tomas Moravec; Helena Plchova; Jitka Folwarczna; Helena Synkova; Helena Ryslava; Viera Ludvikova; Michal Smahel

    2012-03-01

    Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2108-120 epitope were found after both methods of vaccine delivery.

  14. Characterization of C-strain “Riems” TAV-epitope escape variants obtained through selective antibody pressure in cell culture

    Directory of Open Access Journals (Sweden)

    Leifer Immanuel

    2012-04-01

    Full Text Available Abstract Classical swine fever virus (CSFV C-strain “Riems” escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo. It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.

  15. Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

    Directory of Open Access Journals (Sweden)

    Tiago Antônio de Oliveira Mendes

    Full Text Available BACKGROUND: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA. METHODOLOGY: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units, it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI and CL Brener (TcVI clones and Y (TcII strain. FINDINGS AND CONCLUSIONS: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in

  16. Experimental validation of multi-epitope peptides including promising MHC class I- and class II-restricted epitopes of four known Leishmania infantum proteins

    Directory of Open Access Journals (Sweden)

    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.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  18. Human CD4+ T cell epitopes from vaccinia virus induced by vaccination or infection.

    Directory of Open Access Journals (Sweden)

    J Mauricio Calvo-Calle

    2007-10-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    . Using this strategy, we characterized eight BoLA-I molecules, and found the peptide specificity to resemble that of human MHC-I molecules with primary anchors most often at P2 and P9, and occasional auxiliary P1/P3/P5/P6 anchors. We analyzed nine reported CTL epitopes from Theileria parva, and in eight...

  1. Evaluation of a multi-epitope subunit vaccine against avian leukosis virus subgroup J in chickens.

    Science.gov (United States)

    Xu, Qingqing; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Zhao, Xiaomin

    2015-12-01

    The intricate sequence and antigenic variability of avian leukosis virus subgroup J (ALV-J) have led to unprecedented difficulties in the development of vaccines. Much experimental evidence demonstrates that ALV-J mutants have caused immune evasion and pose a challenge for traditional efforts to develop effective vaccines. To investigate the potential of a multi-epitope vaccination strategy to prevent chickens against ALV-J infections, a recombinant chimeric multi-epitope protein X (rCMEPX) containing both immunodominant B and T epitope concentrated domains selected from the major structural protein of ALV-J using bioinformatics approach was expressed in Escherichia coli Rosetta (DE3). Its immunogenicity and protective efficacy was studied in chickens. The results showed that rCMEPX could elicit neutralizing antibodies and cellular responses, and antibodies induced by rCMEPX could specifically recognize host cell naturally expressed ALV-J proteins, which indicated that the rCMEPX is a good immunogen. Challenge experiments showed 80% chickens that received rCMEPX were well protected against ALV-J challenge. This is the first report of a chimeric multi-epitope protein as a potential immunogen against ALV-J.

  2. The Epitope Study on the SARS-CoV Nucleocapsid Protein

    Institute of Scientific and Technical Information of China (English)

    Shuting Li; Xiaolei Li; Jingqiang Wang; Zhengfeng Zhou; Jinxiu Liu; Jianmin Shao; Tingting Lei; Jianqiu Fang; Ningzhi Xu; Siqi Liu; Liang Lin; Hao Wang; Jianning Yin; Yan Ren; Zhe Zhao; Jie Wen; Cuiqi Zhou; Xumin Zhang

    2003-01-01

    The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA),the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins,whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.

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

    Science.gov (United States)

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

    2012-01-01

    Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors. PMID:22405859

  4. Thioreductase containing epitopes inhibit the development of type 1 diabetes in the NOD mouse model

    Directory of Open Access Journals (Sweden)

    Elin eMalek Abrahimians

    2016-03-01

    Full Text Available Autoreactive CD4+ T cells recognizing islet-derived antigens play a primary role in type 1 diabetes. Specific suppression of such cells therefore represents a strategic target for the cure of the disease. We have developed a methodology by which CD4+ T cells acquire apoptosis-inducing properties on antigen-presenting cells after cognate recognition of natural sequence epitopes. We describe here that inclusion of a thiol-disulfide oxidoreductase (thioreductase motif within the flanking residues of a single MHC class II-restricted GAD65 epitope induces GAD65-specific cytolytic CD4+ T cells (cCD4+ T. The latter, obtained either in vitro or by active immunization, acquire an effector memory phenotype and lyse APCs by a Fas-FasL interaction. Further, cCD4+ T cells eliminate by apoptosis activated bystander CD4+ T cells recognizing alternative epitopes processed by the same APC. Active immunization with a GAD65 class II-restricted thioreductase-containing T cell epitope protects mice from diabetes and abrogates insulitis. Passive transfer of in vitro-elicited cCD4+ T cells establishes that such cells are efficient in suppressing autoimmunity. These findings provide strong evidence for a new vaccination strategy to prevent type 1 diabetes.

  5. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø;

    1997-01-01

    . Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  6. 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.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of...

  8. Epitope specificity of human immunodeficiency virus-1 antibody dependent cellular cytotoxicity [ADCC] responses.

    Science.gov (United States)

    Pollara, Justin; Bonsignori, Mattia; Moody, M Anthony; Pazgier, Marzena; Haynes, Barton F; Ferrari, Guido

    2013-07-01

    Antibody dependent cellular cytotoxicity [ADCC] has been suggested to play an important role in control of Human Immunodeficiency Virus-1 [HIV-1] viral load and protection from infection. ADCC antibody responses have been mapped to multiple linear and conformational epitopes within the HIV-1 envelope glycoproteins gp120 and gp41. Many epitopes targeted by antibodies that mediate ADCC overlap with those recognized by antibodies capable of virus neutralization. In addition, recent studies conducted with human monoclonal antibodies derived from HIV-1 infected individuals and HIV-1 vaccine-candidate vaccinees have identified a number of antibodies that lack the ability to capture primary HIV-1 isolates or mediate neutralizing activity, but are able to bind to the surface of infected CD4+ T cells and mediate ADCC. Of note, the conformational changes in the gp120 that may not exclusively relate to binding of the CD4 molecule are important in exposing epitopes recognized by ADCC responses. Here we discuss the HIV-1 envelope epitopes targeted by ADCC antibodies in the context of the potential protective capacities of ADCC. PMID:24191939

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

    Directory of Open Access Journals (Sweden)

    Wu Shipo

    2012-06-01

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

  10. Prediction of Secondary Structure and B Cell Epitope of GH Protein from Acipenser sinensis%中华鲟GH蛋白二级结构和B细胞抗原表位的预测

    Institute of Scientific and Technical Information of China (English)

    刘红艳; 杨东; 张繁荣; 余来宁

    2009-01-01

    [Objective] The aim was to predict the secondary structure and B cell epitope of growth hormone (GH) protein from Acipenser sinensis. [Method] With the amino acid sequence of GH protein from A.sinensis as the base, the secondary structure of GH protein from A.sinensis was predicted by the method of Garnier-Robson, Chou-Fasman and Karplus-Schulz, and its cell epitope was predicted by the method of Kyte-Doolittle, Emini and Jameson-Wolf. [Result] The sections of 18-23, 55-56, 67-73, 83-86, 112-114, 151-157 and 209-211 in the N terminal of GH protein molecule had softer structure and these sections could sway or fold to produce more complex tertiary structure. The sections of 19-23, 51-71, 84-95, 128-139, 164-176 and 189-196 in the N terminal of GH protein could be the epitope of B cell and there were flexible regions in these sections or near these sections of GH protein molecule. So the dominant regions could be in these sections or near these sections. [Conclusion] The research provided the basis for the preparation of monoclonal antibody of GH protein from A.sinensis and provided the reference for the discussion for the molecular regulation mechanism of A.sinensis.

  11. Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes.

    Science.gov (United States)

    Vidotto, O; McElwain, T F; Machado, R Z; Perryman, L E; Suarez, C E; Palmer, G H

    1995-12-01

    Rhoptries are involved in host cell invasion and rhoptry polypeptides, including the Babesia bigemina rhoptry-associated protein-1 (RAP-1), are targets for protective immune responses. Polyclonal antisera produced against isolated rhoptries is directed predominantly against RAP-1 and reacts with both the merozoite and the membrane of parasitized erythrocytes. To determine whether these B cell epitopes associated with the parasitized erythrocyte are derived from RAP-1 or, alternatively, from previously undetected merozoite polypeptides, monoclonal antibodies (mAbs) were generated from mice immunized with rhoptries isolated from the JG-29 clone of the Mexico strain. The anti-RAP-1 mAbs bound only merozoites in a punctate immunofluorescence pattern. A second group of four mAbs, none of which were reactive with RAP-1, bound the parasitized erythrocyte. Two of these latter mAbs, 64/44.17.3 and 64/05.7.2, reacted only with parasitized erythrocytes that had been permeabilized. MAb 64/44.17.3 bound a 54-kDa merozoite polypeptide while 64/05.7.2 bound a > or = 225-kDa merozoite polypeptide. MAbs 64/32.8.5 and 64/38.5.3 recognized epitopes on 17.5- and 76-kDa polypeptides exposed on the external surface of intact parasitized erythrocytes. The results indicate that the identified RAP-1 epitopes are not associated with the erythrocyte cytoskeleton or membrane and that anti-RAP-1 immunity is most likely generated against the free merozoite. All new mAbs reacted with every B. bigemina strain tested (Mexico, Puerto Rico, St. Croix, Texcoco, Jaboticabal). The conservation of RAP-1 epitopes among these strains supports the continued testing of RAP-1 as a vaccine component. In addition, the identification of epitopes expressed on the surface of erythrocytes infected with all five strains provides new candidate immunogens.

  12. Distribution of lipophosphoglycan-associated epitopes in different Leishmania species and in African trypanosomes.

    Science.gov (United States)

    Tolson, D L; Schnur, L F; Jardim, A; Pearson, T W

    1994-01-01

    Monoclonal antibody (mAb) CA7AE binds specifically to the phosphorylated Gal-beta 1,4-Man disaccharide repeat epitope of Leishmania donovani lipophosphoglycan (LPG). This mAb detected the repeat epitope in most but not all of a wide variety of Leishmania species and strains examined. MAb CA7AE also bound to both glycoprotein and carbohydrate antigens in medium from L. donovani promastigote cultures. Specifically, mAb CA7AE bound the delipidated form of LPG, the phosphoglycan, and a glycoprotein both of which are released into the medium by the parasite indicating that both share a specific phosphorylated carbohydrate epitope. The epitope was detected in sera from L. donovani-infected (kala-azar positive) patients when mAb CA7AE was used in an antigen-capture enzyme-linked immunosorbent assay (ELISA). MAb L157 is specific for a protein that is found associated with L. donovani LPG, the lipophosphoglycan-associated protein (LPGAP). This mAb bound to molecules in all 19 strains (representing 9 species) of Leishmania promastigotes and to molecules in 2 species of Trypanosoma procyclic culture forms. This wide distribution of the LPGAP epitope implies that it may have a conserved function, for example, in the biochemistry or arrangement of parasite surface molecules. In addition, since the LPGAP is involved in the stimulation of T lymphocyte proliferation, its wide distribution amongst different Leishmania species suggests that it may be an ideal molecule for testing as a vaccine for leishmaniasis. PMID:7528916

  13. Prediction of promiscuous epitopes in the e6 protein of three high risk human papilloma viruses: a computational approach.

    Science.gov (United States)

    Subramanian, Nirmala; Chinnappan, Sudandiradoss

    2013-01-01

    A najor current challenge and constraint in cervical cancer research is the development of vaccines against human papilloma virus (HPV) epitopes. Although many studies are done on epitope identification on HPVs, no computational work has been carried out for high risk forms which are considered to cause cervical cancer. Of all the high risk HPVs, HPV 16, HPV 18 and HPV 45 are responsible for 94% of cervical cancers in women worldwide. In this work, we computationally predicted the promiscuous epitopes among the E6 proteins of high risk HPVs. We identified the conserved residues, HLA class I, HLA class II and B-cell epitopes along with their corresponding secondary structure conformations. We used extremely precise bioinformatics tools like ClustalW2, MAPPP, NetMHC, EpiJen, EpiTop 1.0, ABCpred, BCpred and PSIPred for achieving this task. Our study identified specific regions 'FAFR(K)DL' followed by 'KLPD(Q)LCTEL' fragments which proved to be promiscuous epitopes present in both human leukocyte antigen (HLA) class I, class II molecules and B cells as well. These fragments also follow every suitable character to be considered as promiscuous epitopes with supporting evidences of previously reported experimental results. Thus, we conclude that these regions should be considered as the important for design of specific therapeutic vaccines for cervical cancer.

  14. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    Science.gov (United States)

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  15. Conformational B-Cell Epitopes Prediction from Sequences Using Cost-Sensitive Ensemble Classifiers and Spatial Clustering

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2014-01-01

    Full Text Available B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction, is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs of 0.721 and 0.703 on two benchmark datasets (bound and unbound using the leave-one-out cross-validation (LOOCV. When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use.

  16. Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

    Science.gov (United States)

    Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use. PMID:25045691

  17. Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

    Science.gov (United States)

    Cariccio, Veronica Lanza; Domina, Maria; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Faleri, Agnese; Bruttini, Marco; Bartolini, Erika; Giuliani, Marzia Monica; Santini, Laura; Brunelli, Brunella; Norais, Nathalie; Borgogni, Erica; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes. PMID:26963435

  18. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

    Science.gov (United States)

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia; Ahlborg, Niklas

    2016-09-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  19. Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    Science.gov (United States)

    Yadav, Pragya; Carr, Matthew T.; Yu, Ruby; Mumbey‐Wafula, Alice

    2016-01-01

    Abstract Introduction The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen‐I (EBNA‐1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA‐1, 3D4, and demonstrated that it cross‐reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA‐1. The aim of the present study was to characterize another antibody to EBNA‐1 that cross‐reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA‐1 that is recognized by these cross‐reactive antibodies. Methods We generated an IgM MAb to EBNA‐1, 16D2, from EBNA‐1 injected mice and demonstrated by ELISA that it cross‐reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA‐1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain. Results Sequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA‐1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA‐1. Conclusion We have demonstrated that two MAbs that bind EBNA‐1 and cross‐react with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE. PMID:27621818

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

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    Salvador Eugenio C. Caoili

    2016-01-01

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

  1. A T Cell Epitope-Based Vaccine Protects Against Chlamydial Infection in HLA-DR4 Transgenic Mice

    Science.gov (United States)

    Li, Weidang; Murthy, Ashlesh K.; Lanka, Gopala Krishna; Chetty, Senthilnath L; Yu, Jieh-Juen; Chambers, James P.; Zhong, Guangming; Forsthuber, Thomas G.; Guentzel, M. Neal; Arulanandam, Bernard P.

    2013-01-01

    Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4+ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4+ T cells in host defense against Chlamydia. In this study, we identify CD4+ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4+ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4+ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4+ clones could be detected in the mouse spleen following C. muridarum and a human C. trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4+ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4+ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model. PMID:24096029

  2. Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98).

    Science.gov (United States)

    Gulati, Upma; Hwang, Chi-Ching; Venkatramani, Lalitha; Gulati, Shelly; Stray, Stephen J; Lee, Janis T; Laver, W Graeme; Bochkarev, Alexey; Zlotnick, Adam; Air, Gillian M

    2002-12-01

    We have characterized monoclonal antibodies raised against the neuraminidase (NA) of a Sydney-like influenza virus (A/Memphis/31/98, H3N2) in a reassortant virus A/NWS/33(HA)-A/Mem/31/98(NA) (H1N2) and nine escape mutants selected by these monoclonal antibodies. Five of the antibodies use the same heavy chain VDJ genes and may not be independent. Another antibody, Mem5, uses the same V(H) and J genes with a different D gene and different isotype. Sequence changes in escape mutants selected by these antibodies occur in two loops of the NA, at amino acid 198, 199, 220, or 221. These amino acids are located on the opposite side of the NA monomer to the major epitopes found in N9 and early N2 NAs. Escape mutants with a change at 198 have reduced NA activity compared to the wild-type virus. Asp198 points toward the substrate binding pocket, and we had previously found that a site-directed mutation of this amino acid resulted in a loss of enzyme activity (M. R. Lentz, R. G. Webster, and G. M. Air, Biochemistry 26:5351-5358, 1987). Mutations at residue 199, 220, or 221 did not alter the NA activity significantly compared to that of wild-type NA. A 3.5-A structure of Mem5 Fab complexed with the Mem/98 NA shows that the Mem5 antibody binds at the sites of escape mutation selected by the other antibodies. PMID:12414967

  3. Computational Analysis of Cysteine Proteases(Clan CA, Family C1)of Leishmania major to Find Potential Epitopic Regions

    Institute of Scientific and Technical Information of China (English)

    Babak Saffari; Hassan Mohabatkar

    2009-01-01

    Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was per-formed by NetCTL program version 1.2. In addition, by using BcePred server, different structural properties of the proteins were predicted to find out their po-tential B cell epitopes. According to this computational analysis, nine regions were predicted as B cell epitopes. The results provide useful information for designing peptide-based vaccines.

  4. Antibodies induced by multi-epitope vaccine showed inhibitory activity against heterologous influenza A virus (H3N2)

    Institute of Scientific and Technical Information of China (English)

    DING Jian; WU Fan; WEI Wei; CHEN Yinghua

    2006-01-01

    In this study, recognition of 4 recombinant viral proteins (GST-NHA1) by the antibodies induced by multi-epitope vaccine was testified. Inhibitory activities of these antibodies were also investigated in vitro against four heterologous influenza A viruses (H3N2). Three epitope-specific antibodies purified by affinity chromatography could reduce the plaque formation. Interestingly, the three neutralizing antibodies in combination showed obvious enhancement of inhibitory activity, suggesting that the development of recombinant multi-epitope vaccine might be an effective way against viral mutation.

  5. Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis.

    Science.gov (United States)

    Jin, Jing; Liss, Nathan M; Chen, Dong-Hua; Liao, Maofu; Fox, Julie M; Shimak, Raeann M; Fong, Rachel H; Chafets, Daniel; Bakkour, Sonia; Keating, Sheila; Fomin, Marina E; Muench, Marcus O; Sherman, Michael B; Doranz, Benjamin J; Diamond, Michael S; Simmons, Graham

    2015-12-22

    We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.

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

    Science.gov (United States)

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

    2013-11-01

    All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus. PMID:24140051

  7. Prediction of a common neutralizing epitope of H5N1 avian influenza virus by in silico molecular docking

    Institute of Scientific and Technical Information of China (English)

    YAN YuanQing; XIA NingShao; LI ShaoWei; YANG ChunYan; LUO WenXin; WANG MingQiao; CHEN YiXin; LUO HaiFeng; WU Ting; ZHANG Jun

    2008-01-01

    The H5N1 avian influenza virus (AIV) has widely spread in Asia, Europe and Africa, making a large amount of economic loss. Recently, our research group has screened a common neutralizing mono-clonal antibody named 8H5, which can neutralize almost all H5 subtype AIV ever isolated so far. Obvi-ously, this monoclonal antibody would benefit for research and development of the universal AIV vac-cine and design of the drug against H5N1 AIV in high mutation rate. In this study, the homology mod-eling was applied to generate the 3D structure of 8H5 Fab fragment, and "canonical structure" method was used to define the specified loop conformation of CDR regions. The model was subjected to en-ergy minimization in cvff force field with Discovery module in Insight Ⅱ program. The resulting model has correct stereochemistry as gauged from the Ramachandran plot calculation and good 3D-structure compatibility as assessed by interaction energy analysis, solvent accessible surface (SAS) analysis, and Profiles-3D approach. Furthermore, the 8H5 Fab model was subjected to docking with three H5 subtype hemagglutinin (HA) structures deposited in PDB (ID No: 1jsm, 2ibx and 2fk0) respectively. The result indicates that the three docked complexes share a common binding interface, but differ in bind-ing angle related with HA structure similarity between viral subtypes. In the light of the three HA inter-faces with structural homology analysis, the common neutralizing epitope on HA recognized by 8H5 consists of 9 incontinuous amino acid residues: Asp68, Asn72, Glu112, Lys113, Ile114, Pro118, Ser120, Tyr137, Tyr252 (numbered as for 1jsm sequence), The primary purpose of the present work is to provide some insight into structure and binding details of a common neutralizing epitope of H5N1 AIV, thereby aiding in the structure-based design of universal AIV vaccines and anti-virus therapeutic drugs.

  8. 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.

  9. Towards universal structure-based prediction of class II MHC epitopes for diverse allotypes.

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    Andrew J Bordner

    Full Text Available The binding of peptide fragments of antigens to class II MHC proteins is a crucial step in initiating a helper T cell immune response. The discovery of these peptide epitopes is important for understanding the normal immune response and its misregulation in autoimmunity and allergies and also for vaccine design. In spite of their biomedical importance, the high diversity of class II MHC proteins combined with the large number of possible peptide sequences make comprehensive experimental determination of epitopes for all MHC allotypes infeasible. Computational methods can address this need by predicting epitopes for a particular MHC allotype. We present a structure-based method for predicting class II epitopes that combines molecular mechanics docking of a fully flexible peptide into the MHC binding cleft followed by binding affinity prediction using a machine learning classifier trained on interaction energy components calculated from the docking solution. Although the primary advantage of structure-based prediction methods over the commonly employed sequence-based methods is their applicability to essentially any MHC allotype, this has not yet been convincingly demonstrated. In order to test the transferability of the prediction method to different MHC proteins, we trained the scoring method on binding data for DRB1*0101 and used it to make predictions for multiple MHC allotypes with distinct peptide binding specificities including representatives from the other human class II MHC loci, HLA-DP and HLA-DQ, as well as for two murine allotypes. The results showed that the prediction method was able to achieve significant discrimination between epitope and non-epitope peptides for all MHC allotypes examined, based on AUC values in the range 0.632-0.821. We also discuss how accounting for peptide binding in multiple registers to class II MHC largely explains the systematically worse performance of prediction methods for class II MHC compared with

  10. Towards universal structure-based prediction of class II MHC epitopes for diverse allotypes.

    Science.gov (United States)

    Bordner, Andrew J

    2010-01-01

    The binding of peptide fragments of antigens to class II MHC proteins is a crucial step in initiating a helper T cell immune response. The discovery of these peptide epitopes is important for understanding the normal immune response and its misregulation in autoimmunity and allergies and also for vaccine design. In spite of their biomedical importance, the high diversity of class II MHC proteins combined with the large number of possible peptide sequences make comprehensive experimental determination of epitopes for all MHC allotypes infeasible. Computational methods can address this need by predicting epitopes for a particular MHC allotype. We present a structure-based method for predicting class II epitopes that combines molecular mechanics docking of a fully flexible peptide into the MHC binding cleft followed by binding affinity prediction using a machine learning classifier trained on interaction energy components calculated from the docking solution. Although the primary advantage of structure-based prediction methods over the commonly employed sequence-based methods is their applicability to essentially any MHC allotype, this has not yet been convincingly demonstrated. In order to test the transferability of the prediction method to different MHC proteins, we trained the scoring method on binding data for DRB1*0101 and used it to make predictions for multiple MHC allotypes with distinct peptide binding specificities including representatives from the other human class II MHC loci, HLA-DP and HLA-DQ, as well as for two murine allotypes. The results showed that the prediction method was able to achieve significant discrimination between epitope and non-epitope peptides for all MHC allotypes examined, based on AUC values in the range 0.632-0.821. We also discuss how accounting for peptide binding in multiple registers to class II MHC largely explains the systematically worse performance of prediction methods for class II MHC compared with those for class I MHC

  11. 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

  12. B-Pred, a structure based B-cell epitopes prediction server

    Directory of Open Access Journals (Sweden)

    Giacò L

    2012-07-01

    Full Text Available Luciano Giacò,1 Massimo Amicosante,2 Maurizio Fraziano,1 Pier Federico Gherardini,1 Gabriele Ausiello,1 Manuela Helmer-Citterich,1 Vittorio Colizzi,1 Andrea Cabibbo11Department of Biology, 2Department of Internal Medicine, University of Rome "Tor Vergata", Rome, ItalyAbstract: The ability to predict immunogenic regions in selected proteins by in-silico methods has broad implications, such as allowing a quick selection of potential reagents to be used as diagnostics, vaccines, immunotherapeutics, or research tools in several branches of biological and biotechnological research. However, the prediction of antibody target sites in proteins using computational methodologies has proven to be a highly challenging task, which is likely due to the somewhat elusive nature of B-cell epitopes. This paper proposes a web-based platform for scoring potential immunological reagents based on the structures or 3D models of the proteins of interest. The method scores a protein's peptides set, which is derived from a sliding window, based on the average solvent exposure, with a filter on the average local model quality for each peptide. The platform was validated on a custom-assembled database of 1336 experimentally determined epitopes from 106 proteins for which a reliable 3D model could be obtained through standard modeling techniques. Despite showing poor sensitivity, this method can achieve a specificity of 0.70 and a positive predictive value of 0.29 by combining these two simple parameters. These values are slightly higher than those obtained with other established sequence-based or structure-based methods that have been evaluated using the same epitopes dataset. This method is implemented in a web server called B-Pred, which is accessible at http://immuno.bio.uniroma2.it/bpred. The server contains a number of original features that allow users to perform personalized reagent searches by manipulating the sliding window's width and sliding step, changing the

  13. Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection.

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    Sunita Gulati

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

  14. Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

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    Agnieszka Korzeniowska-Kowal

    Full Text Available Lipopolysaccharide (LPS, the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia and tumor tissue (ganglioneuroma. Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is

  15. Thermal processing effects on peanut allergen Ara h 2 allergenicity in mice and its antigenic epitope structure.

    Science.gov (United States)

    Zhang, Wenju; Zhu, Qingqing; Zhang, Tong; Cai, Qin; Chen, Qin

    2016-12-01

    Ara h 2 was purified from peanuts that were thermally treated by various processes, including boiling, glycation, frying and roasting. The allergenicity of Ara h 2 in Balb/c mice and the influence of thermal processing on the structural characteristics, and binding capacity of three core antigenic epitopes were studied. The results demonstrated that boiling, glycation and frying induced the down-regulation of the allergenicity of Ara h 2 in Balb/c mice, the collapse of its tertiary/secondary structure, and a reduction in the core epitope binding capacity; roasting showed a comparable allergenicity and the weakest inhibitory effect on core epitope binding capacity. These results indicate that thermal processing causes alteration of the protein structure and core epitopes of Ara h 2, and may affect its allergenicity. PMID:27374581

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

    DEFF Research Database (Denmark)

    Deng, Xiaohong; Zheng, Xuxu; Yang, Huanming;

    2014-01-01

    druggable epitopes/targets. We employed the PROSITE Scan to detect structurally conserved motifs and PRINTS to search for linearly conserved motifs of ECD HER2. We found that the epitopes recognized by trastuzumab and pertuzumab are located in the predicted conserved motifs of ECD HER2, supporting our...... relevant anti-HER2 antibodies. In the present study, we present a novel computational approach as an auxiliary tool for identification of novel HER2 epitopes. We hypothesized that the structurally and linearly evolutionarily conserved motifs of the extracellular domain of HER2 (ECD HER2) contain potential...... initial hypothesis. Considering that structurally and linearly conserved motifs can provide functional specific configurations, we propose that by comparing the two types of conserved motifs, additional druggable epitopes/targets in the ECD HER2 protein can be identified, which can be further modified...

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation......, the latter encompassing 12 different HLA class I supertypes with > 99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-1 interactions were synthesized, tested for peptide-HLA-1 interactions...... in a biochemical assay and for influenza-specific, HLA-1-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-1 binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes...

  18. Protective immunity against Trichinella spiralis infection induced by a multi-epitope vaccine in a murine model.

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    Yuan Gu

    Full Text Available Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87 to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual

  19. Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-1 19 of Plasmodium falciparum.

    Science.gov (United States)

    Omosun, Y O; Adoro, S; Anumudu, C I; Odaibo, A B; Uthiapibull, C; Holder, A A; Nwagwu, M; Nwuba, R I

    2009-03-01

    Merozoite surface protein-1(19) (MSP-1(19)) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-1(19) on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-1(19) vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-1(19) antibodies to modified mutants of MSP-1(19) was analysed by ELISA. The MSP-1(19) mutant proteins with single substitutions at positions 22 (Leu-->Arg), 43 (Glu-->Leu) and 53 (Asn-->Arg) and the MSP-1(19) mutant protein with multiple substitutions at positions 27+31+34+43 (Glu-->Tyr, Leu-->Arg, Tyr-->Ser, Glu-->Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32-80%) with antibodies that bound to the mutants MSP-1(19) containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21-28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-1(19) based malaria vaccines. Although these MSP-1(19) mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-1(19) mutants in the design of a malaria vaccine. PMID:19081386

  20. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

    OpenAIRE

    J Field; Nikawa, J; Broek, D; MacDonald, B.; Rodgers, L; Wilson, I A; Lerner, R A; Wigler, M

    1988-01-01

    We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be ...

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

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

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

  2. The Selection of DNA Aptamers for Two Different Epitopes of Thrombin Was Not Due to Different Partitioning Methods

    OpenAIRE

    Wilson, Robert; Cossins, Andrew; Nicolau, Dan V.; Missailidis, Sotiris

    2013-01-01

    Nearly all aptamers identified so far for any given target molecule have been specific for the same binding site (epitope). The most notable exception to the 1 aptamer per target molecule rule is the pair of DNA aptamers that bind to different epitopes of thrombin. This communication refutes the suggestion that these aptamers exist because different partitioning methods were used when they were selected. The possibility that selection of these aptamers was biased by conflicting secondary stru...

  3. Expression of a repeating phosphorylated disaccharide lipophosphoglycan epitope on the surface of macrophages infected with Leishmania donovani.

    OpenAIRE

    Tolson, D L; Turco, S J; Pearson, T W

    1990-01-01

    Murine peritoneal macrophages were infected with living, virulent Leishmania donovani promastigotes. At intervals after infection, the macrophage surfaces were probed for the expression of lipophosphoglycan (LPG) epitopes by immunofluorescence with anti-LPG monoclonal antibodies. A repeating phosphorylated disaccharide epitope of LPG was detected as early as 5 to 10 min postinfection and was initially localized to the immediate area of internalization of the promastigote into the macrophage. ...

  4. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection

    OpenAIRE

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2014-01-01

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11–21 aa contain EV-71-specific antigenic sites, whereas positions 1–5 and 51–100 contain epitopes shared with hum...

  5. Immunization of rabbits with synthetic peptides derived from a highly conserved β-sheet epitope region underneath the receptor binding site of influenza A virus

    Directory of Open Access Journals (Sweden)

    Ideno S

    2013-11-01

    Full Text Available Shoji Ideno,1,3 Kaoru Sakai,1 Mikihiro Yunoki,2–4 Ritsuko Kubota-Koketsu,3,5 Yuji Inoue,3 Shota Nakamura,6 Teruo Yasunaga,6 Yoshinobu Okuno,5 Kazuyoshi Ikuta3 1Infectious Pathogen Research Section, Central Research Laboratory, Research and Development Division, Japan Blood Products Organization, Kobe, Japan; 2Research and Development Promotion Section, Research and Development Division, Japan Blood Products Organization, Tokyo, Japan; 3Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan; 4Department of Veterinary Microbiology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan; 5Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa, Japan; 6Department of Genome Informatics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Background: There is increasing concern about the speed with which health care providers can administer prophylaxis and treatment in an influenza pandemic. Generally, it takes several months to manufacture an influenza vaccine by propagation of the virus in chicken eggs or cultured cells. Newer, faster protocols for the production of vaccines that induce broad-spectrum immunity are therefore highly desirable. We previously developed human monoclonal antibody B-1 that shows broadly neutralizing activity against influenza A virus H3N2. B-1 recognizes an epitope region that includes an antiparallel β-sheet structure underneath the receptor binding site of influenza hemagglutinin (HA. In this study, the efficacy of a synthetic peptide vaccine derived from this epitope region against influenza A was evaluated. Materials and methods: Two peptides were synthesized, the upper and lower peptides. These peptides comprise amino acid residues 167–187 and 225–241, respectively, of the B-1 epitope region of HA, which is involved in

  6. Identification of a highly immunoreactive epitope of Brugia malayi TPx recognized by the endemic sera.

    Science.gov (United States)

    Madhumathi, Jayaprakasam; Prince, Prabhu Rajaiah; Gayatri, Subash Chellam; Aparnaa, Ramanathan; Kaliraj, Perumal

    2010-12-01

    Filarial thiordoxin peroxidase is a major antioxidant that plays a crucial role in parasite survival. Although Brugia malayi TPx has been shown to be a potential vaccine candidate, it shares 63% homology with its mammalian counterpart, limiting its use as a vaccine or drug target. In silico analysis of TPx sequence revealed a linear B epitope in the host's nonhomologous region. The peptide sequence (TPx peptide(27-48)) was synthesized, and its reactivity with clinical sera from an endemic region was analyzed. The peptide showed significantly high reactivity (P patent infection. The high reactivity of the peptide with endemic immune sera equivalent to that of whole protein shows that it forms a dominant B epitope of TPx protein and thus could be utilized for incorporation into a multiepitope vaccine construct for filariasis. PMID:21158641

  7. 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....

  8. Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes.

    Science.gov (United States)

    Deng, Lei; Linero, Florencia; Saelens, Xavier

    2016-01-01

    Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically contained facilities. We provide protocols for the characterization and purification of recombinant viruslike particles derived from filamentous bacteriophages. As an example, we focus on filamentous Escherichia coli fd phage displaying a conserved influenza A virus epitope that is fused genetically to the N-terminus of the major coat protein of this phage. A step-by-step procedure to obtain a high-titer, pure recombinant phage preparation is provided. We also describe a quality control experiment based on a biological readout of the purified fd phage preparation. These protocols together with the highlighted critical steps may facilitate generic implementation of the provided procedures for the display of other epitopes by recombinant fd phages.

  9. 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

    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...

  10. Design and Antigenic Epitopes Prediction of a New Trial Recombinant Multiepitopic Rotaviral Vaccine: In Silico Analyses.

    Science.gov (United States)

    Jafarpour, Sima; Ayat, Hoda; Ahadi, Ali Mohammad

    2015-01-01

    Rotavirus is the major etiologic factor of severe diarrheal disease. Natural infection provides protection against subsequent rotavirus infection and diarrhea. This research presents a new vaccine designed based on computational models. In this study, three types of epitopes are considered-linear, conformational, and combinational-in a proposed model protein. Several studies on rotavirus vaccines have shown that VP6 and VP4 proteins are good candidates for vaccine production. In the present study, a fusion protein was designed as a new generation of rotavirus vaccines by bioinformatics analyses. This model-based study using ABCpred, BCPREDS, Bcepred, and Ellipro web servers showed that the peptide presented in this article has the necessary properties to act as a vaccine. Prediction of linear B-cell epitopes of peptides is helpful to investigate whether these peptides are able to activate humoral immunity.

  11. 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

    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......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...... 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...

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

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby

    2007-01-01

    The spread of existing infectious diseases and the emergence of new ones call for efficient methods for vaccine development. Some of the important players in conferring immunity against pathogens are the Cytotoxic T Lymphocytes (CTL), which eliminate infected cells. Due to their deleterious effects...... vaccine design as well as for diagnostic purposes and is the centre of focus of this thesis: Part I of the thesis is an introduction to the field. In part II, I describe how we generated a method, NetCTL, for predicting CTL epitopes by integrating existing methods for predicting proteasomal cleavage, TAP......: 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...

  13. Design and Antigenic Epitopes Prediction of a New Trial Recombinant Multiepitopic Rotaviral Vaccine: In Silico Analyses.

    Science.gov (United States)

    Jafarpour, Sima; Ayat, Hoda; Ahadi, Ali Mohammad

    2015-01-01

    Rotavirus is the major etiologic factor of severe diarrheal disease. Natural infection provides protection against subsequent rotavirus infection and diarrhea. This research presents a new vaccine designed based on computational models. In this study, three types of epitopes are considered-linear, conformational, and combinational-in a proposed model protein. Several studies on rotavirus vaccines have shown that VP6 and VP4 proteins are good candidates for vaccine production. In the present study, a fusion protein was designed as a new generation of rotavirus vaccines by bioinformatics analyses. This model-based study using ABCpred, BCPREDS, Bcepred, and Ellipro web servers showed that the peptide presented in this article has the necessary properties to act as a vaccine. Prediction of linear B-cell epitopes of peptides is helpful to investigate whether these peptides are able to activate humoral immunity. PMID:25965449

  14. 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

    2016-03-01

    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. PMID:26430814

  15. Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Liu, Lifeng; Qian, Suhong; Chen, Huanchun; Li, Xiangmin

    2014-12-01

    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. PMID:25490764

  16. Epitope fluctuations in the human papillomavirus are under dynamic allosteric control: a computational evaluation of a new vaccine design strategy.

    Science.gov (United States)

    Singharoy, Abhishek; Polavarapu, Abhigna; Joshi, Harshad; Baik, Mu-Hyun; Ortoleva, Peter

    2013-12-11

    The dynamic properties of the capsid of the human papillomavirus (HPV) type 16 were examined using classical molecular dynamics simulations. By systematically comparing the structural fluctuations of the capsid protein, a strong dynamic allosteric connection between the epitope containing loops and the h4 helix located more than 50 Å away is identified, which was not recognized thus far. Computer simulations show that restricting the structural fluctuations of the h4 helix is key to rigidifying the epitopes, which is thought to be required for eliciting a proper immune response. The allostery identified in the components of the HPV is nonclassical because the mean structure of the epitope carrying loops remains unchanged, but as a result of allosteric effect the structural fluctuations are altered significantly, which in turn changes the biochemical reactivity profile of the epitopes. Exploiting this novel insight, a new vaccine design strategy is proposed wherein a relatively small virus capsid fragment is deposited on a silica nanoparticle in such a way that the fluctuations of the h4 helix are suppressed. The structural and dynamic properties of the epitope carrying loops on this hybrid nanoparticle match the characteristics of epitopes found on the full virus-like particle precisely, suggesting that these nanoparticles may serve as potent, cost-effective, and safe alternatives to traditionally developed vaccines. The structural and dynamic properties of the hybrid nanoparticle are examined in detail to establish the general concepts of the proposed new design. PMID:24199651

  17. Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells

    Directory of Open Access Journals (Sweden)

    Li Jinyao

    2011-05-01

    Full Text Available Abstract Background We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40low IL-10high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg. However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. Results In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. Conclusions Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

  18. 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.

  19. 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

    2016-03-01

    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.

  20. 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.

  1. Stratification of responders towards eculizumab using a structural epitope mapping strategy

    DEFF Research Database (Denmark)

    Volk, Anna-Luisa; Hu, Francis Jingxin; Berglund, Magnus M.;

    2016-01-01

    for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we...... treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics....

  2. Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.

    Science.gov (United States)

    Bhatt, Jay M; Styers, Melanie L; Sztul, Elizabeth

    2016-01-01

    Before the advent of molecular methods to tag proteins, visualization of proteins within cells required the use of antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Epitope tagging allows the detection of all proteins with existing sequence information, irrespective of the availability of antibodies directed against them. This technique involves the generation of DNA constructs that express the protein of interest tagged with an epitope that can be recognized by a commercially available antibody. Proteins can be tagged with a wide variety of epitopes using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into mammalian cell lines and, in most cases, tightly mimic the behavior of the endogenous protein. Tagged proteins exogenously expressed in cells provide different types of information depending on the subsequent detection approaches. Using immunofluorescence and immunoelectron microscopy with anti-tag antibodies, relative to known markers of cellular organelles, can provide information on the subcellular localization of the tagged protein and may provide clues regarding the protein's function. Immunofluorescence with anti-tag antibodies can also be utilized to assess the tagged protein's responses to cellular signals and pharmacological treatments. Immunoprecipitations with anti-tag antibodies can recover protein complexes containing the protein of interest, resulting in the identification of interacting proteins. Recovery of tagged proteins on affinity matrices allows their purification for use in biochemical assays. In addition, specialized fluorescent tags, such as the green fluorescent protein (GFP) allow the analysis of cellular dynamics in live cells in real time. PMID:27515071

  3. Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift

    OpenAIRE

    Cantor, Jason R.; Yoo, Tae Hyeon; Dixit, Aakanksha; Iverson, Brent L.; Forsthuber, Thomas G.; Georgiou, George

    2011-01-01

    A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve ext...

  4. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

    Directory of Open Access Journals (Sweden)

    George Janssen

    Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Phase Variation in H Type I and Lewis a Epitopes of Helicobacter pylori Lipopolysaccharide

    OpenAIRE

    Appelmelk, Ben J; Martino, M. Celeste; Veenhof, Eveline; Monteiro, Mario A.; Maaskant, Janneke J.; Negrini, Riccardo; Lindh, Frank; Perry, Malcolm; Del Giudice, Giuseppe; Vandenbroucke-Grauls, Christina M. J. E.

    2000-01-01

    Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Lex), Ley, and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Lex and Ley and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a...

  7. The Utilization and Limitation of CD133 Epitopes in Lung Cancer Stem Cells Research

    OpenAIRE

    Chen, Yin; Hong ZHONG

    2011-01-01

    Lung cancer is one of the most common tumor, which lacks of effective clinical treatment to lead to desirable prognosis. According to cancer stem cell hypothesis, lung cancer stem cells are considered to be responsible for carcinogenesis, development, metastasis, recurrence, invasion, resistance to chemotherapy and radiotherapy of lung cancer. In recent years, more and more institutes used glycosylated CD133 epitopes to define, isolate, purify lung cancer stem cells. However, along with deepl...

  8. B-Pred, a structure based B-cell epitopes prediction server.

    Science.gov (United States)

    Giacò, Luciano; Amicosante, Massimo; Fraziano, Maurizio; Gherardini, Pier Federico; Ausiello, Gabriele; Helmer-Citterich, Manuela; Colizzi, Vittorio; Cabibbo, Andrea

    2012-01-01

    The ability to predict immunogenic regions in selected proteins by in-silico methods has broad implications, such as allowing a quick selection of potential reagents to be used as diagnostics, vaccines, immunotherapeutics, or research tools in several branches of biological and biotechnological research. However, the prediction of antibody target sites in proteins using computational methodologies has proven to be a highly challenging task, which is likely due to the somewhat elusive nature of B-cell epitopes. This paper proposes a web-based platform for scoring potential immunological reagents based on the structures or 3D models of the proteins of interest. The method scores a protein's peptides set, which is derived from a sliding window, based on the average solvent exposure, with a filter on the average local model quality for each peptide. The platform was validated on a custom-assembled database of 1336 experimentally determined epitopes from 106 proteins for which a reliable 3D model could be obtained through standard modeling techniques. Despite showing poor sensitivity, this method can achieve a specificity of 0.70 and a positive predictive value of 0.29 by combining these two simple parameters. These values are slightly higher than those obtained with other established sequence-based or structure-based methods that have been evaluated using the same epitopes dataset. This method is implemented in a web server called B-Pred, which is accessible at http://immuno.bio.uniroma2.it/bpred. The server contains a number of original features that allow users to perform personalized reagent searches by manipulating the sliding window's width and sliding step, changing the exposure and model quality thresholds, and running sequential queries with different parameters. The B-Pred server should assist experimentalists in the rational selection of epitope antigens for a wide range of applications. PMID:22888263

  9. Scope for using plant viruses to present epitopes from animal pathogens.

    Science.gov (United States)

    Porta; Lomonossoff

    1998-01-01

    Epitope presentation to the immune system for vaccination purposes can be achieved either via an inactivated or attenuated form of a pathogen or via its isolated antigenic sequences. When free, these peptides can adopt a variety of conformations, most of which will not exist in their native environment. Conjugation to carrier proteins restricts mobility of the peptides and increases their immunogenicity. A high local concentration of epitopes boosts the immune response further and can be generated by the use of self-aggregating carriers, such as the capsid proteins of viruses. In this regard plant viruses have in recent years started to make an impact as safer alternatives to the use of bacterial and attenuated animal viruses: the latter both require propagation in costly cell-culture systems where they can undergo reversion towards a virulent form and/or become contaminated by other pathogens. Plant virus-based vectors can be multiplied cheaply and to high yields (exceeding 1 mg/g plant tissue) in host plants. Both helical (tobacco mosaic virus, potato virus X, alfalfa mosaic virus) and icosahedral (cowpea mosaic virus, tomato bushy stunt virus) particles have been used to express a number of animal B-cell epitopes, whose immunogenic properties have been explored to varying degrees. Copyright 1998 John Wiley & Sons, Ltd. PMID:10398492

  10. De Novo Transcriptome Analysis of Allium cepa L. (Onion Bulb to Identify Allergens and Epitopes.

    Directory of Open Access Journals (Sweden)

    Hemalatha Rajkumar

    Full Text Available 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.

  11. Human CD8(+) T Cells Target Multiple Epitopes in Respiratory Syncytial Virus Polymerase.

    Science.gov (United States)

    Burbulla, Daniel; Günther, Patrick S; Peper, Janet K; Jahn, Gerhard; Dennehy, Kevin M

    2016-06-01

    Respiratory syncytial virus (RSV) infection is a serious health problem in young children, immunocompromised patients, and the elderly. The development of novel prevention strategies, such as a vaccine to RSV, is a high priority. One strategy is to design a peptide-based vaccine that activates appropriate CD8(+) T-cell responses. However, this approach is limited by the low number of RSV peptide epitopes defined to date that activate CD8(+) T cells. We aimed to identify peptide epitopes that are presented by common human leukocyte antigen types (HLA-A*01, -A*02, and -B*07). We identify one novel HLA-A*02-restricted and two novel HLA-A*01-restricted peptide epitopes from RSV polymerase. Peptide-HLA multimer staining of specific T cells from healthy donor peripheral blood mononuclear cell, the memory phenotype of such peptide-specific T cells ex vivo, and functional IFNγ responses in short-term stimulation assays suggest that these peptides are recognized during RSV infection. Such peptides are candidates for inclusion into a peptide-based RSV vaccine designed to stimulate defined CD8(+) T-cell responses.

  12. Molecular Design and Immunogenicity of a Multiple-epitope FMDV Antigen and DNA Vaccination

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This article reports the design and construction of a multiple-epitope foot and mouth disease virus (FMDV)antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-γ and cytotoxic activity from mice immunized with pA were significantly increased. pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asia1 FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.

  13. Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies.

    Science.gov (United States)

    Tsiantoulas, Dimitrios; Perkmann, Thomas; Afonyushkin, Taras; Mangold, Andreas; Prohaska, Thomas A; Papac-Milicevic, Nikolina; Millischer, Vincent; Bartel, Caroline; Hörkkö, Sohvi; Boulanger, Chantal M; Tsimikas, Sotirios; Fischer, Michael B; Witztum, Joseph L; Lang, Irene M; Binder, Christoph J

    2015-02-01

    Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.

  14. The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin.

    Science.gov (United States)

    Shcherbinin, D N; Alekseeva, S V; Shmarov, M M; Smirnov, Yu A; Naroditskiy, B S; Gintsburg, A L

    2016-01-01

    Vaccination has been successfully used to prevent influenza for a long time. Influenza virus hemagglutinin (HA), which induces a humoral immune response in humans and protection against the flu, is the main antigenic component of modern influenza vaccines. However, new seasonal and pandemic influenza virus variants with altered structures of HA occasionally occur. This allows the pathogen to avoid neutralization with antibodies produced in response to previous vaccination. Development of a vaccine with the new variants of HA acting as antigens takes a long time. Therefore, during an epidemic, it is important to have passive immunization agents to prevent and treat influenza, which can be monoclonal or single-domain antibodies with universal specificity (broad-spectrum agents). We considered antibodies to conserved epitopes of influenza virus antigens as universal ones. In this paper, we tried to characterize the main B-cell epitopes of hemagglutinin and analyze our own and literature data on broadly neutralizing antibodies. We conducted a computer analysis of the best known conformational epitopes of influenza virus HAs using materials of different databases. The analysis showed that the core of the HA molecule, whose antibodies demonstrate pronounced heterosubtypic activity, can be used as a target for the search for and development of broad-spectrum antibodies to the influenza virus. PMID:27099781

  15. Amino acid residues 56 to 69 of HLA-A2 specify an antigenic determinant shared by HLA-A2 and HLA-B17.

    Science.gov (United States)

    Ways, J P; Rothbard, J B; Parham, P

    1986-07-01

    The mouse monoclonal antibody MA2.1 was previously used to define an epitope shared by native HLA-A2 and HLA-B17 molecules and amino acid sequence comparison of nine HLA-A,B,C molecules identified residues 62 to 65 as the region most likely to form this epitope. An unabsorbed rabbit antiserum raised against a peptide corresponding to residues 56 to 69 of HLA-A2 gives highly specific reactions with HLA-A2 and HLA-B17 heavy chains in Western blots. No interactions with native HLA-A2 and B17 molecules were detected in a variety of assays. Although the topographic relationship between the epitopes recognized by the rabbit antiserum and the monoclonal antibody could not be determined, the results show that residues 56 to 69 of HLA-A2 can form epitopes with specificity for HLA-A2 and HLA-B17.

  16. 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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    , suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library......Background: Development and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational...... epitopes. Objective: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. Methods: Polyclonal sera from three individual patients...

  18. Epitope Spreading of Autoantibody Response to PLA2R Associates with Poor Prognosis in Membranous Nephropathy.

    Science.gov (United States)

    Seitz-Polski, Barbara; Dolla, Guillaume; Payré, Christine; Girard, Christophe A; Polidori, Joel; Zorzi, Kevin; Birgy-Barelli, Eléonore; Jullien, Perrine; Courivaud, Cécile; Krummel, Thierry; Benzaken, Sylvia; Bernard, Ghislaine; Burtey, Stéphane; Mariat, Christophe; Esnault, Vincent L M; Lambeau, Gérard

    2016-05-01

    The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFRPLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.

  19. An integrative approach to CTL epitope prediction: A combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

    DEFF Research Database (Denmark)

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

    2005-01-01

    Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have al.......The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php....

  20. Phase I trial of an alhydrogel adjuvanted hepatitis B core virus-like particle containing epitopes of Plasmodium falciparum circumsporozoite protein.

    Directory of Open Access Journals (Sweden)

    Aric L Gregson

    Full Text Available UNLABELLED: The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP(3] epitope, an HLA-restricted CD4 (NANPNVDPNANP epitope and a universal T cell epitope (T* (amino acids 326-345, NF54 isolate. We assessed an Alhydrogel (aluminum hydroxide-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29-75% and 29-63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty