Science.gov

Sample records for conserved linear epitopes

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

    PubMed

    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

  2. Fine level epitope mapping and conservation analysis of two novel linear B-cell epitopes of the avian infectious bronchitis coronavirus nucleocapsid protein.

    PubMed

    Han, Zongxi; Zhao, Fei; Shao, Yuhao; Liu, Xiaoli; Kong, Xiangang; Song, Yang; Liu, Shengwang

    2013-01-01

    The nucleocapsid (N) protein of the infectious bronchitis virus (IBV) may play an essential role in the replication and translation of viral RNA. The N protein can also induce high titers of cross-reactive antibodies and cell-mediated immunity, which protects chickens from acute infection. In this study, we generated two monoclonal antibodies (mAbs), designated as 6D10 and 4F10, which were directed against the N protein of IBV using the whole viral particles as immunogens. Both of the mAbs do not cross react with Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV) and subtype H9 avian influenza virus (AIV). After screening a phage display peptide library and peptide scanning, we identified two linear B-cell epitopes that were recognized by the mAbs 6D10 and 4F10, which corresponded to the amino acid sequences (242)FGPRTK(247) and (195)DLIARAAKI(203), respectively, in the IBV N protein. Alignments of amino acid sequences from a large number of IBV isolates indicated that the two epitopes, especially (242)FGPRTK(247), were well conserved among IBV strains. This conclusion was further confirmed by the relationships of 18 heterologous sequences to the 2 mAbs. The novel mAbs and the epitopes identified will be useful for developing diagnostic assays for IBV infections. PMID:23123213

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

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

  5. Identification of a conserved linear epitope using a monoclonal antibody against non-structural protein 3B of foot-and-mouth disease virus.

    PubMed

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

    2016-02-01

    Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae that has caused severe economic losses in many countries of the world. Regular vaccinations have been effectively used to control foot-and-mouth disease (FMD) in countries where the disease is enzootic. Distinguishing between infected and vaccinated animals in herds after immunization is an important component of effective eradication strategies. Nonstructural protein (NSP) 3B of FMDV is part of a larger antigen that is used for this differential diagnosis. In this study, an FMDV serotype-independent monoclonal antibody (MAb) against NSP 3B, 5D12, was generated. Using western blot, it was revealed that MAb 5D12 binds to three fragments of 3B displaying the motifs G(1)PYAGPLERQKPLK(14), K(18)LPQQEGPYAGPMER(32) and V(45)KEGPYEGPVKKPVA(59). The motif G(1)PYAGPLERQKPLK(14) was chosen for further mapping. Different truncated motifs derived from the motif G(1)PYAGPLERQKPLK(14) were expressed as GST-fusion constructs for western blot analysis. The results showed that the 5-aa peptide P(2)YAGP(6) was the minimal epitope reactive to MAb 5D12. Subsequent alanine-scanning mutagenesis analysis revealed that Pro(2), Gly(5) and Pro(6) were crucial for MAb 5D12 binding to P(2)YAGP(6). Furthermore, through sequence alignment analysis, the epitope PxxGP recognized by 5D12 was found to be present not only in 3B-1 but also in 3B2 and 3B3 and was highly conserved in seven serotypes of FMDV strains. Western blot analysis also revealed that the peptide epitope could be recognized by sera from FMDV-infected pigs and cattle. Thus, the 5D12-recognized 3B epitope identified here provides theoretical support for the development of MAb 5D12 as a differential diagnosis reagent for FMDV infection. PMID:26563318

  6. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  7. A novel linear neutralizing epitope of hepatitis E virus.

    PubMed

    Tang, Zi-Min; Tang, Ming; Zhao, Min; Wen, Gui-Ping; Yang, Fan; Cai, Wei; Wang, Si-Ling; Zheng, Zi-Zheng; Xia, Ning-Shao

    2015-07-01

    Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459-606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423-437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine. PMID:26051517

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

    PubMed

    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

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

    PubMed Central

    Birkelund, S; Larsen, B; Holm, A; Lundemose, A G; Christiansen, G

    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 part of the HSP70 molecule known as connect II. Images PMID:7513310

  10. Identification of a Novel Conserved B Cell Epitope in the N Protein of Equine Arteritis Virus (Bucyrus Strain).

    PubMed

    Chen, Jie; Guo, Xinggang; Li, Lianwei

    2015-09-01

    The nucleocapsid (N) protein is the most conserved structural protein in equine arteritis virus (EAV). This study aimed to identify the minimal conserved B cell epitope on the EAV N protein. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay. Moreover, 11 overlapping peptides (named MBP-N1 to MBP-N11) were designed to localize the linear antigenic epitope within the N protein. The peptides were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot. The minimal conserved B cell epitope on the EAV N protein was identified. The homology analysis was also performed. An EAV N-reactive mAb was selected and designated as 1C11. Indirect ELISA results showed that overlapping domain between MBP-N10 and MBP-N11 was recognized by the mAb 1C11. Furthermore, the indirect ELISA and Western blot showed that (101)QRKVAP(106) was the minimal linear epitope of the EAV N protein. The homology analysis showed that the identified epitope was conserved among all EAV strains analyzed in this work, with the exception of the ARVAC. One EAV N-specific mAb (1C11) was developed, and a minimal linear peptide epitope ((101)QRKVAP(106)) within the N protein was identified. PMID:26331346

  11. Cross-reactive, linear B cell epitopes of the influenza virus matrix protein 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Evaluate antibody responses to the conserved influenza matrix protein. Background: Little is known about the B cell epitopes in conserved internal influenza proteins or their role in viral immunity and immunopathogenesis. Based on epitope information present in the Immune Epitope Database...

  12. Comparative Analysis of Evolutionarily Conserved Motifs of Epidermal Growth Factor Receptor 2 (HER2) Predicts Novel Potential Therapeutic Epitopes

    PubMed Central

    Deng, Xiaohong; Zheng, Xuxu; Yang, Huanming; Moreira, José Manuel Afonso; Brünner, Nils; Christensen, Henrik

    2014-01-01

    Overexpression of human epidermal growth factor receptor 2 (HER2) is associated with tumor aggressiveness and poor prognosis in breast cancer. With the availability of therapeutic antibodies against HER2, great strides have been made in the clinical management of HER2 overexpressing breast cancer. However, de novo and acquired resistance to these antibodies presents a serious limitation to successful HER2 targeting treatment. The identification of novel epitopes of HER2 that can be used for functional/region-specific blockade could represent a central step in the development of new clinically 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 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 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 for potential therapeutic application. Thus, this novel computational process for predicting or searching for potential epitopes or key target sites may contribute to epitope-based vaccine and function-selected drug design, especially when x-ray crystal structure protein data is not available. PMID:25192037

  13. T cell epitope redundancy: cross-conservation of the TCR face between pathogens and self and its implications for vaccines and autoimmunity.

    PubMed

    Moise, Leonard; Beseme, Sarah; Tassone, Ryan; Liu, Rui; Kibria, Farzana; Terry, Frances; Martin, William; De Groot, Anne S

    2016-05-01

    T cells are extensively trained on 'self' in the thymus and then move to the periphery, where they seek out and destroy infections and regulate immune response to self-antigens. T cell receptors (TCRs) on T cells' surface recognize T cell epitopes, short linear strings of amino acids presented by antigen-presenting cells. Some of these epitopes activate T effectors, while others activate regulatory T cells. It was recently discovered that T cell epitopes that are highly conserved on their TCR face with human genome sequences are often associated with T cells that regulate immune response. These TCR-cross-conserved or 'redundant epitopes' are more common in proteins found in pathogens that have co-evolved with humans than in other non-commensal pathogens. Epitope redundancy might be the link between pathogens and autoimmune disease. This article reviews recently published data and addresses epitope redundancy, the "elephant in the room" for vaccine developers and T cell immunologists. PMID:26588466

  14. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    SciTech Connect

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

    2009-05-21

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

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

    PubMed Central

    2016-01-01

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

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

    PubMed

    Caoili, Salvador Eugenio C

    2016-01-01

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

  17. An ELISA detecting antibody to conserved pestivirus epitopes.

    PubMed

    Paton, D J; Ibata, G; Edwards, S; Wensvoort, G

    1991-01-01

    A monoclonal antibody based competition-ELISA is described for the detection of pestivirus antibodies directed against conserved epitopes on the p80 viral protein. The ELISA detected increases in serum antibody following experimentally induced infections of pigs, cattle and sheep with a wide range of pestiviruses, although the sensitivity of the test was not uniform for the different viruses studied. The ELISA was compared with virus neutralization tests for the assessment of porcine, bovine and ovine field sera. At a cut-off value of 50% inhibition, the ELISA showed a high specificity relative to virus neutralization tests, but appeared less sensitive for the detection of some weakly positive samples from pigs. Sera from both ruminants and pigs could be assessed without any modification of the test. PMID:1713919

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

    PubMed

    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

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

    PubMed

    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

  20. Identification of a highly conserved and surface exposed B-cell epitope on the nucleoprotein of influenza A virus.

    PubMed

    Gui, Xun; Ge, Pinghui; Wang, Xuliang; Yang, Kunyu; Yu, Hai; Zhao, Qinjian; Chen, Yixin; Xia, Ningshao

    2014-06-01

    Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope. PMID:24136709

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

    PubMed Central

    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

  2. [Study on the B cell linear epitopes of rabies virus CVS-11 nucleoprotein].

    PubMed

    Lv, Xin-Jun; Shen, Xin-Xin; Yu, Peng-Cheng; Li, Hao; Wang, Li-Hua; Tang, Qing; Liang, Guo-Dong

    2014-05-01

    To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes. PMID:25118379

  3. Fine-Mapping of Immunodominant Linear B-Cell Epitopes of the Staphylococcus Aureus SEB Antigen Using Short Overlapping Peptides

    PubMed Central

    Zhao, Zhuo; Li, Bin; Sun, He-Qiang; Zhang, Jin-Yong; Wang, Yi-Lin; Chen, Li; Hu, Jian; He, Ya-Fei; Zeng, Hao; Zou, Quan-Ming; Wu, Chao

    2014-01-01

    Staphylococcal enterotoxin B (SEB) is one of the most potent Staphylococcus aureus exotoxins (SEs). Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB) and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97–114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257) were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97–112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97–112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen. PMID:24599257

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

    PubMed

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

    2016-10-01

    Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein. PMID:27438076

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

    PubMed Central

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

    2015-01-01

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression. PMID:26293607

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

    PubMed

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

    2015-01-01

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression. PMID:26293607

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

    PubMed Central

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

    2016-01-01

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

  8. HLA Preferences for Conserved Epitopes: A Potential Mechanism for Hepatitis C Clearance

    PubMed Central

    Rao, Xiangyu; Hoof, Ilka; van Baarle, Debbie; Keşmir, Can; Textor, Johannes

    2015-01-01

    Hepatitis C virus (HCV) infections affect more than 170 million people worldwide. Most of these individuals are chronically infected, but some clear the infection rapidly. Host factors seem to play a key role in HCV clearance, among them are the human leukocyte antigen (HLA) class I molecules. Certain HLA molecules, e.g., B*27 and B*57, are associated with viral clearance. To identify potential mechanisms for these associations, we assess epitope distribution differences between HLA molecules using experimentally verified and in silico predicted HCV epitopes. Specifically, we show that the NS5B protein harbors the largest fraction of conserved regions among all HCV proteins. Such conserved regions could be good targets for cytotoxic T-cell (CTL) responses. We find that the protective HLA-B*27 molecule preferentially presents cytotoxic T-cell (CTL) epitopes from NS5B and, in general, presents the most strongly conserved epitopes among the 23 HLA molecules analyzed. In contrast, HLA molecules known to be associated with HCV persistence do not have similar preferences and appear to target the variable P7 protein. Overall, our analysis suggests that by targeting highly constrained – and thereby conserved – regions of HCV, the protective HLA molecule HLA-B*27 reduces the ability of HCV to escape the cytotoxic T-cell response of the host. For visualizing the distribution of both experimentally verified and predicted epitopes across the HCV genome, we created the HCV epitope browser, which is available at theory.bio.uu.nl/ucqi/hcv. PMID:26579127

  9. Measles viruses of genotype H1 evade recognition by vaccine-induced neutralizing antibodies targeting the linear haemagglutinin noose epitope.

    PubMed

    Finsterbusch, Tim; Wolbert, Anne; Deitemeier, Ingrid; Meyer, Kerstin; Mosquera, Maria Mar; Mankertz, Annette; Santibanez, Sabine

    2009-11-01

    The linear haemagglutinin noose epitope (HNE; aa 379-410) is a protective B-cell epitope and considered to be highly conserved in both the vaccine and the wild-type measles virus (MeV) haemagglutinin (H) proteins. Vaccine virus-derived monoclonal antibodies (mAbs) BH6 and BH216, which target the HNE, neutralized MeVs of genotypes B3, C2, D4, D5, D6, D7 and D8, and the vaccine strain Edmonston Zagreb. In the case of genotype H1, only strain Berlin.DEU/44.01 was neutralized by these mAbs, whereas strains Shenyang.CHN/22.99 and Sofia.BGR/19.05 were not. The H gene sequences of these two strains showed an exchange of proline 397 (P397) to leucine (L397). Mutated H proteins, with P397 exchanged to L and vice versa, were compared with original H proteins by indirect fluorescence assay. H proteins exhibiting P397 but not those with L397 were recognized by BH6 and BH216. This indicates that L397 leads to the loss of the neutralizing HNE. In contrast, human sera obtained from vaccinees (n=10) did not discriminate between genotype H1 variants P397 and L397. This concurs with the epidemiological observation that the live-attenuated vaccine protects against both H1 variants. Furthermore, we demonstrated that MeVs of genotype H1 also lack the neutralizing epitopes defined by the vaccine virus-induced mAbs BH15, BH125 and BH47. The loss of several neutralizing epitopes, as shown for H1 viruses currently circulating endemically in Asia, implies that epitope monitoring should be considered to be included in measles surveillance. PMID:19625457

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

    PubMed

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

    2012-12-14

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    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

  13. Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

    PubMed Central

    Nascimento, Eduardo J. M.; Mailliard, Robbie B.; Khan, Asif M.; Sidney, John; Sette, Alessandro; Guzman, Nicole; Paulaitis, Michael; de Melo, Andréa Barbosa; Cordeiro, Marli T.; Gil, Laura V. G.; Lemonnier, Françoir; Rinaldo, Charles; August, J. Thomas; Marques, Ernesto T. A.

    2013-01-01

    Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. PMID:24130917

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

    PubMed

    Khrustalev, Vladislav Victorovich

    2009-01-01

    We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ). PMID:19811425

  15. Characterization of an Isotype-Dependent Monoclonal Antibody against Linear Neutralizing Epitope Effective for Prophylaxis of Enterovirus 71 Infection

    PubMed Central

    Khong, Wei Xin; Yan, Benedict; Premanand, Balraj; Alonso, Sylvie; Chow, Vincent T. K.; Kwang, Jimmy

    2012-01-01

    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. PMID:22279543

  16. Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine: HLA-A3-restricted GAIA vaccine epitopes.

    PubMed

    De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T; Skowron, Gail; Mayer, Kenneth H; Buus, Soren; Boyle, Christine M; Martin, William D

    2012-07-01

    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 of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs. PMID:22777092

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

    PubMed

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

    2015-02-18

    Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5-24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin. PMID:25597941

  18. Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

    PubMed Central

    Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

    2014-01-01

    ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular

  19. Elicitation from virus-naive individuals of cytotoxic T lymphocytes directed against conserved HIV-1 epitopes

    PubMed Central

    Reche, Pedro A; Keskin, Derin B; Hussey, Rebecca E; Ancuta, Petronela; Gabuzda, Dana; Reinherz, Ellis L

    2006-01-01

    Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNγ producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects. PMID:16674822

  20. Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen▿

    PubMed Central

    Kelly-Cirino, Cassandra D.; Mantis, Nicholas J.

    2009-01-01

    The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines. PMID:19703971

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

    PubMed Central

    2012-01-01

    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-neutralizing epitope, spanning

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

    PubMed

    Kunwar, Pratima; Hawkins, Natalie; Dinges, Warren L; Liu, Yi; Gabriel, Erin E; Swan, David A; Stevens, Claire E; Maenza, Janine; Collier, Ann C; Mullins, James I; Hertz, Tomer; Yu, Xuesong; Horton, Helen

    2013-01-01

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

  3. Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

    PubMed Central

    Kunwar, Pratima; Hawkins, Natalie; Dinges, Warren L.; Liu, Yi; Gabriel, Erin E.; Swan, David A.; Stevens, Claire E.; Maenza, Janine; Collier, Ann C.; Mullins, James I.; Hertz, Tomer; Yu, Xuesong; Horton, Helen

    2013-01-01

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

  4. Using random forest to classify linear B-cell epitopes based on amino acid properties and molecular features.

    PubMed

    Huang, Jian-Hua; Wen, Ming; Tang, Li-Juan; Xie, Hua-Lin; Fu, Liang; Liang, Yi-Zeng; Lu, Hong-Mei

    2014-08-01

    Identification and characterization of B-cell epitopes in target antigens was one of the key steps in epitopes-driven vaccine design, immunodiagnostic tests, and antibody production. Experimental determination of epitopes was labor-intensive and expensive. Therefore, there was an urgent need of computational methods for reliable identification of B-cell epitopes. In current study, we proposed a novel peptide feature description method which combined peptide amino acid properties with chemical molecular features. Based on these combined features, a random forest (RF) classifier was adopted to classify B-cell epitopes and non-epitopes. RF is an ensemble method that uses recursive partitioning to generate many trees for aggregating the results; and it always produces highly competitive models. The classification accuracy, sensitivity, specificity, Matthews correlation coefficient (MCC), and area under the curve (AUC) values for current method were 78.31%, 80.05%, 72.23%, 0.5836, and 0.8800, respectively. These results showed that an appropriate combination of peptide amino acid features and chemical molecular features with a RF model could enhance the prediction performance of linear B-cell epitopes. Finally, a freely online service was available at http://sysbio.yznu.cn/Research/Epitopesprediction.aspx. PMID:24721579

  5. Wealth redistribution in conservative linear kinetic models

    NASA Astrophysics Data System (ADS)

    Toscani, G.

    2009-10-01

    We introduce and discuss kinetic models for wealth distribution which include both taxation and uniform redistribution. The evolution of the continuous density of wealth obeys a linear Boltzmann equation where the background density represents the action of an external subject on the taxation mechanism. The case in which the mean wealth is conserved is analyzed in full details, by recovering the analytical form of the steady states. These states are probability distributions of convergent random series of a special structure, called perpetuities. Among others, Gibbs distribution appears as steady state in case of total taxation and uniform redistribution.

  6. Identification of a novel linear B-cell epitope in the UL26 and UL26.5 proteins of Duck Enteritis Virus

    PubMed Central

    2010-01-01

    Background The Unique Long 26 (UL26) and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV), which is an unassigned member of the family Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c), which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb). The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV. Results A mAb (designated 1C8) was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA) and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, 520IYYPGE525, which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The 520IYYPGE525 motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif 521YYPGE525 in the epitope sequence was conserved among the alphaherpesviruses. Conclusion A mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was 520IYYPGE525. The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV. PMID:20836860

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

    PubMed

    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

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

    PubMed Central

    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

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

    PubMed

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

    2013-11-01

    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

  10. Identification of three PPV1 VP2 protein-specific B cell linear epitopes using monoclonal antibodies against baculovirus-expressed recombinant VP2 protein.

    PubMed

    Sun, Jianhui; Huang, Liping; Wei, Yanwu; Wang, Yiping; Chen, Dongjie; Du, Wenjuan; Wu, Hongli; Feng, Li; Liu, Changming

    2015-11-01

    Porcine parvovirus type 1 (PPV1) is a major causative agent of embryonic and fetal death in swine. The PPV1 VP2 protein is closely associated with viral immunogenicity for eliciting neutralizing antibodies, but its antigenic structures have been largely unknown. We generated three monoclonal antibodies (MAbs) against baculovirus-expressed recombinant PPV1 VP2 protein. A PEPSCAN analysis identified the minimal B cell linear epitopes of PPV1 VP2 based on these MAbs. Three core epitopes, (228)QQITDA(233), (284)RSLGLPPK(291), and (344)FEYSNGGPFLTPI(356), were defined and mapped onto three-dimensional models of the PPV1 virion and VP2 monomer. The epitope (228)QQITDA(233) is exposed on the virion surface, and the other two are located inside the protein. An alignment of the PPV1 VP2 amino acid sequences showed that (284)RSLGLPPK(291) and (344)FEYSNGGPFLTPI(356) are absolutely conserved, whereas (228)QQITDA(233) has a single substitution at residue 233 in some (S → A or T). We developed a VP2 epitope-based indirect enzyme-linked immunosorbent assay (iELISA) to test for anti-PPV1 antibodies. In a comparative analysis with an immunoperoxidase monolayer assay using 135 guinea pig sera, the VP2-epitope-based iELISA had a concordance rate of 85.19 %, sensitivity of 83.33 %, and specificity of 85.47 %. MAb 8H6 was used to monitor VP2 during the PPV1 replication cycle in vitro with an indirect immunofluorescence assay, which indicated that newly encapsulated virions are released from the nucleus at 24 h postinfection and the PPV1 replication cycle takes less than 24 h. This study provides valuable information clarifying the antigenic structure of PPV1 VP2 and lays the foundations for PPV1 serodiagnosis and antigen detection. PMID:26153140

  11. Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding*

    PubMed Central

    Serup Andersen, Olga; Boisguerin, Prisca; Glerup, Simon; Skeldal, Sune; Volkmer, Rudolf; Willnow, Thomas E.; Nykjær, Anders; Andersen, Olav M.

    2010-01-01

    Sortilin acts as a cell surface receptor for pro-neurotrophins (pro-NT) that upon complex formation with the p75 neurotrophin receptor (p75NTR) is able to signal neuronal cell death. Here we screened a sortilin peptide library comprising 16-mer overlapping sequences for binding of the pro-domains of nerve growth factor and brain-derived neurotrophic factor. We find that a linear surface-exposed sequence, 163RIFRSSDFAKNF174, constitutes an important pro-NT binding epitope in sortilin. Systematic mutational analysis revealed residues Arg163, Phe165, Arg166, and Phe170 to be critical for the interaction. Expression of a sortilin mutant in which these four amino acids were substituted by alanines disrupted pro-NT binding without affecting receptor heterodimerization with p75NTR or binding of ligands that selectively engages the centrally located tunnel in the β-propeller of sortilin. We furthermore demonstrate that a peptide comprising the ligand-binding epitope can prevent pro-NT-induced apoptosis in RN22 schwannoma cells. PMID:20159974

  12. Identification and Validation of a Linear Protective Neutralizing Epitope in the β-Pore Domain of Alpha Toxin

    PubMed Central

    Oscherwitz, Jon; Cease, Kemp B.

    2015-01-01

    The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha

  13. Identification and validation of a linear protective neutralizing epitope in the β-pore domain of alpha toxin.

    PubMed

    Oscherwitz, Jon; Cease, Kemp B

    2015-01-01

    The plethora of virulence factors associated with Staphylococcus aureus make this bacterium an attractive candidate for a molecularly-designed epitope-focused vaccine. This approach, which necessitates the identification of neutralizing epitopes for incorporation into a vaccine construct, is being evaluated for pathogens where conventional approaches have failed to elicit protective humoral responses, like HIV-1 and malaria, but may also hold promise for pathogens like S. aureus, where the elicitation of humoral immunity against multiple virulence factors may be required for development of an effective vaccine. Among the virulence factors employed by S. aureus, animal model and epidemiological data suggest that alpha toxin, a multimeric β-pore forming toxin like protective antigen from Bacillus anthracis, is particularly critical, yet no candidate neutralizing epitopes have been delineated in alpha toxin to date. We have previously shown that a linear determinant in the 2β2-2β3 loop of the pore forming domain of B. anthracis protective antigen is a linear neutralizing epitope. Antibody against this site is highly potent for neutralizing anthrax lethal toxin in vitro and for protection of rabbits in vivo from virulent B. anthracis. We hypothesized that sequences in the β-pore of S. aureus alpha toxin that share structural and functional homology to β-pore sequences in protective antigen would contain a similarly critical neutralizing epitope. Using an in vivo mapping strategy employing peptide immunogens, an optimized in vitro toxin neutralization assay, and an in vivo dermonecrosis model, we have now confirmed the presence of this epitope in alpha toxin, termed the pore neutralizing determinant. Antibody specific for this determinant neutralizes alpha toxin in vitro, and is highly effective for mitigating dermonecrosis and bacterial growth in a mouse model of S. aureus USA300 skin infection. The delineation of this linear neutralizing determinant in alpha

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

    PubMed

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

    2016-01-01

    Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine. PMID:26895191

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

    PubMed Central

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

    2016-01-01

    Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine. PMID:26895191

  16. Polyclonal B Cell Responses to Conserved Neutralization Epitopes in a Subset of HIV-1-Infected Individuals▿†

    PubMed Central

    Tomaras, Georgia D.; Binley, James M.; Gray, Elin S.; Crooks, Emma T.; Osawa, Keiko; Moore, Penny L.; Tumba, Nancy; Tong, Tommy; Shen, Xiaoying; Yates, Nicole L.; Decker, Julie; Wibmer, Constantinos Kurt; Gao, Feng; Alam, S. Munir; Easterbrook, Philippa; Abdool Karim, Salim; Kamanga, Gift; Crump, John A.; Cohen, Myron; Shaw, George M.; Mascola, John R.; Haynes, Barton F.; Montefiori, David C.; Morris, Lynn

    2011-01-01

    A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 “tier 2” viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein. PMID:21849452

  17. Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects

    PubMed Central

    Roff, Shannon R; Sanou, Missa P; Rathore, Mobeen H; Levy, Jay A; Yamamoto, Janet K

    2015-01-01

    Cross-reactive peptides on HIV-1 and FIV p24 protein sequences were studied using peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected long-term survivors (LTS; >10 y of infection without antiretroviral therapy, ART), short-term HIV-1 infected subjects not on ART, and ART-treated HIV-1 infected subjects. IFNγ-ELISpot and CFSE-proliferation analyses were performed with PBMC using overlapping HIV-1 and FIV p24 peptides. Over half of the HIV-1 infected subjects tested (22/31 or 71%) responded to one or more FIV p24 peptide pools by either IFNγ or T-cell proliferation analysis. PBMC and T cells from infected subjects in all 3 HIV+ groups predominantly recognized one FIV p24 peptide pool (Fp14) by IFNγ production and one additional FIV p24 peptide pool (Fp9) by T-cell proliferation analysis. Furthermore, evaluation of overlapping SIV p24 peptide sequences identified conserved epitope(s) on the Fp14/Hp15-counterpart of SIV, Sp14, but none on Fp9-counterpart of SIV, Sp9. The responses to these FIV peptide pools were highly reproducible and persisted throughout 2–4 y of monitoring. Intracellular staining analysis for cytotoxins and phenotyping for CD107a determined that peptide epitopes from Fp9 and Fp14 pools induced cytotoxic T lymphocyte-associated molecules including perforin, granzyme B, granzyme A, and/or expression of CD107a. Selected FIV and corresponding SIV epitopes recognized by HIV-1 infected patients indicate that these protein sequences are evolutionarily conserved on both SIV and HIV-1 (e.g., Hp15:Fp14:Sp14). These studies demonstrate that comparative immunogenicity analysis of HIV-1, FIV, and SIV can identify evolutionarily-conserved T cell-associated lentiviral epitopes, which could be used as a vaccine for prophylaxis or immunotherapy. PMID:25844718

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

    PubMed Central

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

    2013-01-01

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

  19. Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins.

    PubMed

    Robotham, Jason M; Hoffman, Gregg G; Teuber, Suzanne S; Beyer, Kirsten; Sampson, Hugh A; Sathe, Shridhar K; Roux, Kenneth H

    2009-09-01

    Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues. Linear IgE-epitope mapping was performed by solid-phase overlapping 15-amino acid peptides probed with IgE from pooled allergic human sera. Several walnut (Jug r 4) and hazelnut (Cor a 9) 11S globulin peptides with reactivity to patient IgE were identified. Comparative alignment with cashew (Ana o 2), peanut (Ara h 3), and soybean G1 (Gly m 6.0101) and G2 (Gly m 6.0201) allergenic homologues revealed several shared allergenic 'hot spots'. Homology modeling was performed based on the atomic structure of the soybean glycinin. Surface map comparisons between the tree nut and peanut homologues revealed structural motifs that could be important for IgE elicitation and binding and show that, contrary to predictions, the reactive epitopes are widely distributed throughout the monomeric subunits, both internally and externally, including regions occluded by quaternary subunit association. These findings reveal structural features that may be important to allergenicity and cross-reactivity of this protein class. PMID:19631385

  20. An Improved Method for Predicting Linear B-cell Epitope Using Deep Maxout Networks.

    PubMed

    Lian, Yao; Huang, Ze Chi; Ge, Meng; Pan, Xian Ming

    2015-06-01

    To establish a relation between an protein amino acid sequence and its tendencies to generate antibody response, and to investigate an improved in silico method for linear B-cell epitope (LBE) prediction. We present a sequence-based LBE predictor developed using deep maxout network (DMN) with dropout training techniques. A graphics processing unit (GPU) was used to reduce the training time of the model. A 10-fold cross-validation test on a large, non-redundant and experimentally verified dataset (Lbtope_Fixed_ non_redundant) was performed to evaluate the performance. DMN-LBE achieved an accuracy of 68.33% and an area under the receiver operating characteristic curve (AUC) of 0.743, outperforming other prediction methods in the field. A web server, DMN-LBE, of the improved prediction model has been provided for public free use. We anticipate that DMN-LBE will be beneficial to vaccine development, antibody production, disease diagnosis, and therapy. PMID:26177908

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  3. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  4. MONOCLONAL ANTIBODIES IDENTIFY CONSERVED EPITOPES ON THE POLYHEDRIN OF 'HELIOTHIS ZEA' NUCLEAR POLYHEDROSIS VIRUS

    EPA Science Inventory

    Recent advances in monoclonal antibody techniques have provided an opportunity to simplify the procedures of serological identification of microorganisms. Because monoclonal antibodies are raised against individual antigenic determinants (epitopes), they can be used to screen wit...

  5. Calnexin induces expansion of antigen-specific CD4(+) T cells that confer immunity to fungal ascomycetes via conserved epitopes.

    PubMed

    Wüthrich, Marcel; Brandhorst, Tristan T; Sullivan, Thomas D; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A; Jenkins, Marc K; Klein, Bruce

    2015-04-01

    Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  6. Calnexin induces expansion of antigen-specific CD4+ T cells that confer immunity to fungal ascomycetes via conserved epitopes

    PubMed Central

    Wüthrich, Marcel; Brandhorst, Tristan T.; Sullivan, Thomas D.; Filutowicz, Hanna; Sterkel, Alana; Stewart, Douglas; Li, Mengyi; Lerksuthirat, Tassanee; LeBert, Vanessa; Shen, Zu Ting; Ostroff, Gary; Deepe, George S.; Hung, Chiung Yu; Cole, Garry; Walter, Jennifer A.; Jenkins, Marc K.; Klein, Bruce

    2015-01-01

    Fungal infections remain a threat due to the lack of broad spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown, but conserved antigen. Using transgenic CD4+ T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats induces expansion of calnexin-specific CD4+ T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4+ T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogeneticity and conservation of calnexin make this fungal protein a promising vaccine target. PMID:25800545

  7. Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays.

    PubMed

    Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus

    2013-01-01

    We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level. PMID:23894373

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

    PubMed Central

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

    2013-01-01

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

  9. A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody

    PubMed Central

    Chain, Benny; Arnold, Jack; Akthar, Samia; Brandt, Michael; Davis, David; Noursadeghi, Mahdad; Lapp, Thabo; Ji, Changhua; Sankuratri, Surya; Zhang, Yanjing; Govada, Lata; Saridakis, Emmanuel; Chayen, Naomi

    2015-01-01

    The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution. PMID:26030924

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

    PubMed

    Dam, Catharina E; Houen, Gunnar; Hansen, Paul R; Trier, Nicole H

    2014-09-01

    Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular, the two aspartate residues were found to be essential for antibody reactivity since these amino acids could not be substituted without a reduction in antibody reactivity. The majority of the remaining amino acids could be substituted without reducing antibody reactivity notably. These results confirm 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. PMID:24792370

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

    PubMed

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

    2016-10-15

    Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. PMID:27590430

  12. Identification of a novel linear epitope in tetanus toxin recognized by a protective monoclonal antibody: implications for vaccine design.

    PubMed

    Luo, Ping; Qin, Liyan; Mao, Xuhu; Chen, Li; Yu, Shu; Li, Qian; Liu, Wei; Zhang, Weijun; Gu, Jiang; Zou, Quanming

    2012-10-01

    Tetanus, a severe infectious disease, is caused by tetanus toxin (TT) from Clostridium tetani, which remains one of the most critical unsolved health problems despite preventive strategies. The carboxyl terminal of TT (TTC) is responsible for the binding of TT to neurons and for its toxicity and has been proven to be immunogenic and protective in various forms. It would therefore be extremely interesting to identify the epitope on TTC at a molecular level. In this study, we generated a neutralizing monoclonal antibody, 5C4, which inhibited TT binding to its receptor and was efficiently protective at 73.7 IU/mg. Moreover, 5C4 recognized a novel linear epitope on TT, namely TC((1155-1171)), which spans from Lys1155 to Val1171. In addition, TC((1155-1171)) was shown to elicit the production of a serum IgG that protected mice against a challenge with TT. These results suggested that TC((1155-1171)) and the monoclonal antibody 5C4 are good candidates for the development of epitope-based vaccines and therapeutic antibodies against tetanus. PMID:22889825

  13. Identification and characterization of immunodominant linear epitopes on the antigenic region of a serine protease in newborn Trichinella larvae.

    PubMed

    Yang, Y; Vallée, I; Lacour, S A; Boireau, P; Cheng, S P; Liu, M Y

    2016-03-01

    An immunodominant serine protease of Trichinella spiralis named NBL1 showed encouraging potential in early diagnosis of trichinellosis in pigs and elicited protective immune responses during infection of animals. To further define serological reagents for diagnostic use, the specific epitopes on NBL protein recognized by the antibody responses of different susceptible hosts need to be defined. The present study described comprehensive mapping of immunodominant linear epitopes in the antigenic region (NBL-C, the C-terminal part of the protein) using various serum samples obtained from three kinds of hosts - pig, wild boar and mice. We identified six peptides which were commonly recognized by sera from pigs experimentally infected with Trichinella and pigs immunized with rNBL1-C; five and four peptides were recognized by sera from wild boars and mice infected with Trichinella, respectively. Three peptides (NBL1-6, -7 and -9) were commonly recognized by antisera in all three hosts, which share the sequence PSSGSRPTYP. We also found that one peptide (NBL1-12) was only recognized by antibodies from pigs immunized with rNBL1-C. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of subunit vaccines and diagnostic tools for trichinellosis. PMID:25989815

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

    PubMed

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

    2010-07-13

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains. PMID:20615945

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

    PubMed Central

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

    2010-01-01

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG+ memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains. PMID:20615945

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

    PubMed Central

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

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

    PubMed

    Lomonte, Bruno

    2012-10-01

    Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer. PMID:22677805

  18. Energy conserving, linear scaling Born-Oppenheimer molecular dynamics.

    PubMed

    Cawkwell, M J; Niklasson, Anders M N

    2012-10-01

    Born-Oppenheimer molecular dynamics simulations with long-term conservation of the total energy and a computational cost that scales linearly with system size have been obtained simultaneously. Linear scaling with a low pre-factor is achieved using density matrix purification with sparse matrix algebra and a numerical threshold on matrix elements. The extended Lagrangian Born-Oppenheimer molecular dynamics formalism [A. M. N. Niklasson, Phys. Rev. Lett. 100, 123004 (2008)] yields microcanonical trajectories with the approximate forces obtained from the linear scaling method that exhibit no systematic drift over hundreds of picoseconds and which are indistinguishable from trajectories computed using exact forces. PMID:23039583

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

    PubMed Central

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

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

  20. Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

    PubMed Central

    Jurkiewicz, E; Hunsmann, G; Schäffner, J; Nisslein, T; Lüke, W; Petry, H

    1997-01-01

    The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological

  1. Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1-VP2 junction regions.

    PubMed Central

    Saikawa, T; Anderson, S; Momoeda, M; Kajigaya, S; Young, N S

    1993-01-01

    Presentation of linear epitopes of the B19 parvovirus capsid proteins as peptides might be a useful vaccine strategy. We produced overlapping fusion proteins to span the viral capsid sequence, inoculated rabbits, and determined whether the resulting antisera contained antibodies that neutralized the ability of the virus to infect human erythroid progenitor cells. Antibodies that bound to virus in an enzyme-linked immunosorbent assay were present in antisera raised against 10 of 11 peptides; strongest activity was found for antisera against the carboxyl-terminal half of the major capsid protein. However, strong neutralizing activity was elicited in animals immunized with peptides from the amino-terminal portion of the unique region of the minor capsid protein and peptides containing the sequence of the junction region between the minor and major capsid proteins. The development of neutralizing activity in animals was elicited most rapidly with the fusion peptide from the first quarter of the unique region. A 20-amino-acid region of the unique region of the minor capsid protein was shown to contain a neutralizing epitope. Multiple antigenic peptides, based on the sequence of the unique region and produced by covalent linkage through a polylysine backbone, elicited strong neutralizing antibody responses. Synthetic peptides and fusion proteins containing small regions of the unique portion of the minor capsid protein might be useful as immunogens in a human vaccine against B19 parvovirus. Images PMID:7684458

  2. General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

    PubMed

    Mendes, T M; Guimarães-Okamoto, P T C; Machado-de-Avila, R A; Oliveira, D; Melo, M M; Lobato, Z I; Kalapothakis, E; Chávez-Olórtegui, C

    2015-06-01

    This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified. PMID:25817000

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

    PubMed

    Xi, Jun; Yan, Huili

    2016-10-01

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

  4. A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

    PubMed Central

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

    2013-01-01

    Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection. PMID:24049169

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

    PubMed Central

    Rosendahl Huber, S. K.; Camps, M. G. M.; Jacobi, R. H. J.; Mouthaan, J.; van Dijken, H.; van Beek, J.; Ossendorp, F.; de Jonge, J.

    2015-01-01

    Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks. PMID:26046664

  6. Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

    PubMed Central

    Méchin, M C; Rousset, E; Girardeau, J P

    1996-01-01

    As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was

  7. On resonance in linear conservation laws without source terms

    NASA Technical Reports Server (NTRS)

    Gingold, H.; Trutzer, V.

    1990-01-01

    A phenomena is described occuring in wave propagation for systems of conservation laws which are not hyperbolic. The nature of the wave propagation for linear systems of conservation laws which are degenerate in the sense that they are not equivalent to diagonal systems is examined. It is concluded that in the degenerate case, 'resonance' occurs, that is, the solutions are combinations of traveling waves and 'resonance waves'. Here resonance means that the solution may become unbounded even if the initial values are bounded. The solutions (waves) can be described as a superposition of 'packets' (groups) of 'resonance waves' traveling with the same speed. These are linear systems with constant coefficients which are self-exciting, which is what one would expect to encounter in nonlinear systems.

  8. Study of immunogenicity of recombinant proteins based on hemagglutinin and neuraminidase conservative epitopes of Influenza A virus

    PubMed Central

    Dukhovlinov, Ilya; Al-Shekhadat, Ruslan; Fedorova, Ekaterina; Stepanova, Ludmila; Potapchuk, Marina; Repko, Irina; Rusova, Olga; Orlov, Anton; Tsybalova, Ludmila; Kiselev, Oleg

    2013-01-01

    Background Recombinant hemagglutinin (rHA) and neurominidase (rNA) developed in our investigation are amino acid sequence consensus variants of H1N1 2009 subtype influenza virus strain, also including immunogenic epitopes typical for other influenza virus subtypes (H3N1 and H5N1). Substitutions were made: typical for Russian virus isolates (in HA – S220T, NA – D248N) and in active centers of molecules – R118L, R293L, R368L; C92S, C417S to increase recombinant proteins stability in E. coli. The aim of the present work was to study immunogenicity of the obtained rHA and rNA. Material/Methods Fragments aa 83–469 of NA and aa 61–287 of HA were chosen because they include the main B-cell epitopes and are the minimal structures for correct folding of target proteins. The designed nucleotide sequences were synthesized and purified and the expression of rNA and rNA were analyzed. For immunization and virus challenge we used influenza viruses A/California/04/2009 (H1N1), A/PR/8/34 (H1N1), A/Perth/16/2009 (H3N2), A/Chicken/Kurgan/05/2005 R.G. (H5N1), and B/Florida/04/2006. Specific IgG levels were determined by ELISA in 96-well ELISA plates. Significant differences of survival in mouse groups were analyzed by Mantel-Cox (log-rank) and Gehan-Breslow-Wilcoxon tests. Results The obtained results demonstrate the high immunogenicity and ability of indicated proteins mixture to provide similar cross-protection against influenza viruses of the H1N1 subtype. Conclusions The data obtained suggest efficient pluripotent vaccine creation based on HA and NA conservative regions. PMID:23969554

  9. Characterization of Protective Epitopes in a Highly Conserved Plasmodium falciparum Antigenic Protein Containing Repeats of Acidic and Basic Residues

    PubMed Central

    Sharma, Pawan; Kumar, Anil; Singh, Balwan; Bharadwaj, Ashima; Sailaja, V. Naga; Adak, T.; Kushwaha, Ashima; Malhotra, Pawan; Chauhan, V. S.

    1998-01-01

    The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasite Plasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparum malaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum. PMID:9596765

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

    PubMed

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

    2014-04-01

    Feline infectious peritonitis virus (FIP virus: FIPV) causes a fatal disease in wild and domestic cats. The development of an FIP-preventive vaccine requires an antigen that does not induce antibody-dependent enhancement, and T helper (Th)1 activity plays an important role in protect against FIPV infection. In the present study, we identified synthetic peptides including Th1 and a linear immunodominant antibody-binding epitope in the S1 domain and M protein of FIPV. We also identified peptides that strongly induce Th1 activity from those derived from the structural proteins (S, M, and N proteins) of FIPV based on this and previous studies (Satoh et al. [19]). No Th1 epitope-containing peptide was identified in the peptides derived from the S1 domain of type I FIPV. In contrast, 7 Th1 epitope-containing peptides were identified in the S1 domain of type II FIPV, and no linear immunodominant antibody-binding epitope was contained in any of these peptides. Eleven Th1 epitope-containing peptides common to each serotype were identified in the M protein-derived peptides, and 2 peptides (M-11 and M-12) contained the linear immunodominant antibody-binding epitope. Of the peptides derived from the S, M, and N proteins of FIPV, those that induced significantly stronger Th1 activity than that of the FIPV antigen were rescreened, and 4 peptides were identified. When 3 of these peptides (M-9, I-S2-15, and II-S1-24) were selected and administered with CpG-ODNs to SPF cats, M-9 and II-S1-24 induced Th1 activity. Our results may provide important information for the development of a peptide-based vaccine against FIPV infection. PMID:24530149

  11. In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9

    PubMed Central

    Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Singh, Balwan; Jiang, Jianlin; Meyer, Esmeralda V. S.; Santos, Fátima; Banic, Dalma Maria; Moreno, Alberto; Galinski, Mary R.; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa

    2016-01-01

    Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and

  12. In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9.

    PubMed

    Rodrigues-da-Silva, Rodrigo Nunes; Martins da Silva, João Hermínio; Singh, Balwan; Jiang, Jianlin; Meyer, Esmeralda V S; Santos, Fátima; Banic, Dalma Maria; Moreno, Alberto; Galinski, Mary R; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa

    2016-01-01

    Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and

  13. Split diversity in constrained conservation prioritization using integer linear programming

    PubMed Central

    Chernomor, Olga; Minh, Bui Quang; Forest, Félix; Klaere, Steffen; Ingram, Travis; Henzinger, Monika; von Haeseler, Arndt

    2015-01-01

    Phylogenetic diversity (PD) is a measure of biodiversity based on the evolutionary history of species. Here, we discuss several optimization problems related to the use of PD, and the more general measure split diversity (SD), in conservation prioritization. Depending on the conservation goal and the information available about species, one can construct optimization routines that incorporate various conservation constraints. We demonstrate how this information can be used to select sets of species for conservation action. Specifically, we discuss the use of species' geographic distributions, the choice of candidates under economic pressure, and the use of predator–prey interactions between the species in a community to define viability constraints. Despite such optimization problems falling into the area of NP hard problems, it is possible to solve them in a reasonable amount of time using integer programming. We apply integer linear programming to a variety of models for conservation prioritization that incorporate the SD measure. We exemplarily show the results for two data sets: the Cape region of South Africa and a Caribbean coral reef community. Finally, we provide user-friendly software at http://www.cibiv.at/software/pda. PMID:25893087

  14. Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.

    PubMed Central

    Redlich, P N; Hoeprich, P D; Colby, C B; Grossberg, S E

    1991-01-01

    The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN-beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta. Images PMID:1708891

  15. B and T Cell Epitope-Based Peptides Predicted from Evolutionarily Conserved and Whole Protein Sequences of Ebola Virus as Vaccine Targets.

    PubMed

    Yasmin, T; Nabi, A H M Nurun

    2016-05-01

    Ebola virus (EBV) has become a serious threat to public health. Different approaches were applied to predict continuous and discontinuous B cell epitopes as well as T cell epitopes from the sequence-based and available three-dimensional structural analyses of each protein of EBV. Peptides '(79) VPSATKRWGFRSGVPP(94) ' from GP1 and '(515) LHYWTTQDEGAAIGLA(530) ' from GP2 of Ebola were found to be the consensus peptidic sequences predicted as linear B cell epitope of which the latter contains a region (519) TTQDEG(524) that fulfilled all the criteria of accessibility, hydrophilicity, flexibility and beta turn region for becoming an ideal B cell epitope. Different nonamers as T cell epitopes were obtained that interacted with different numbers of MHC class I and class II alleles with a binding affinity of <100 nm. Interestingly, these alleles also bound to the MHC class I alleles mostly prevalent in African and South Asian regions. Of these, 'LANETTQAL' and 'FLYDRLAST' nonamers were predicted to be the most potent T cell epitopes and they, respectively, interacted with eight and twelve class I alleles that covered 63.79% and 54.16% of world population, respectively. These nonamers were found to be the core sequences of 15mer peptides that interacted with the most common class II allele, HLA-DRB1*01:01. They were further validated for their binding to specific class I alleles using docking technique. Thus, these predicted epitopes may be used as vaccine targets against EBV and can be validated in model hosts to verify their efficacy as vaccine. PMID:26939891

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

    PubMed

    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

  17. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

    PubMed Central

    Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R.; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. PMID:27494183

  18. Antibodies to a single, conserved epitope in Anopheles APN1 inhibit universal transmission of Plasmodium falciparum and Plasmodium vivax malaria.

    PubMed

    Armistead, Jennifer S; Morlais, Isabelle; Mathias, Derrick K; Jardim, Juliette G; Joy, Jaimy; Fridman, Arthur; Finnefrock, Adam C; Bagchi, Ansu; Plebanski, Magdalena; Scorpio, Diana G; Churcher, Thomas S; Borg, Natalie A; Sattabongkot, Jetsumon; Dinglasan, Rhoel R

    2014-02-01

    Malaria transmission-blocking vaccines (TBVs) represent a promising approach for the elimination and eradication of this disease. AnAPN1 is a lead TBV candidate that targets a surface antigen on the midgut of the obligate vector of the Plasmodium parasite, the Anopheles mosquito. In this study, we demonstrated that antibodies targeting AnAPN1 block transmission of Plasmodium falciparum and Plasmodium vivax across distantly related anopheline species in countries to which malaria is endemic. Using a biochemical and immunological approach, we determined that the mechanism of action for this phenomenon stems from antibody recognition of a single protective epitope on AnAPN1, which we found to be immunogenic in murine and nonhuman primate models and highly conserved among anophelines. These data indicate that AnAPN1 meets the established target product profile for TBVs and suggest a potential key role for an AnAPN1-based panmalaria TBV in the effort to eradicate malaria. PMID:24478095

  19. Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

    PubMed Central

    Weiland, Emilie; Bolz, Sylvia; Weiland, Frank; Herbst, Werner; Raamsman, Martin J. B.; Rottier, Peter J. M.; De Vries, Antoine A. F.

    2000-01-01

    We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171–186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (GL). Two of the EAV GL-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-GL MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The GL-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-GL MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-GL MAbs. PMID

  20. Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification.

    PubMed

    Cassels, F J; Deal, C D; Reid, R H; Jarboe, D L; Nauss, J L; Carter, J M; Boedeker, E C

    1992-06-01

    Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and

  1. Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification.

    PubMed Central

    Cassels, F J; Deal, C D; Reid, R H; Jarboe, D L; Nauss, J L; Carter, J M; Boedeker, E C

    1992-01-01

    Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and

  2. Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

    PubMed Central

    Chen, Chien-Wen; Wu, Ming-Shan; Huang, Yi-Jen; Cheng, Chao-An; Chang, Chi-Yao

    2015-01-01

    Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development. PMID:25938761

  3. Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses

    PubMed Central

    Wan, Hongquan; Gao, Jin; Xu, Kemin; Chen, Hongjun; Couzens, Laura K.; Rivers, Katie H.; Easterbrook, Judy D.; Yang, Kevin; Zhong, Lei; Rajabi, Mohsen; Ye, Jianqiang; Sultana, Ishrat; Wan, Xiu-Feng; Liu, Xiufan; Perez, Daniel R.; Taubenberger, Jeffery K.

    2013-01-01

    Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza. PMID:23785204

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-05-15

    The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1-/-, or Myd88-/- mice. Comparison of gnotobiotic Rag1-/- mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. PMID:25795776

  6. Characterizing the Interactions between a Naturally Primed Immunoglobulin A and Its Conserved Bacteroides thetaiotaomicron Species-specific Epitope in Gnotobiotic Mice*

    PubMed Central

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

    2015-01-01

    The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1−/−, or Myd88−/− mice. Comparison of gnotobiotic Rag1−/− mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. PMID:25795776

  7. Conserved epitopes on the hemagglutinin-neuraminidase proteins of human and bovine parainfluenza type 3 viruses: nucleotide sequence analysis of variants selected with monoclonal antibodies.

    PubMed Central

    Coelingh, K J; Winter, C C; Murphy, B R; Rice, J M; Kimball, P C; Olmsted, R A; Collins, P L

    1986-01-01

    We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains. Images PMID:2427750

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-01

    A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination. PMID:27009612

  11. The Crystal Structure of PPIL1 Bound to Cyclosporine A Suggests a Binding Mode for a Linear Epitope of the SKIP Protein

    PubMed Central

    Stegmann, Christian M.; Lührmann, Reinhard; Wahl, Markus C.

    2010-01-01

    Background The removal of introns from pre-mRNA is carried out by a large macromolecular machine called the spliceosome. The peptidyl-prolyl cis/trans isomerase PPIL1 is a component of the human spliceosome and binds to the spliceosomal SKIP protein via a binding site distinct from its active site. Principal Findings Here, we have studied the PPIL1 protein and its interaction with SKIP biochemically and by X-ray crystallography. A minimal linear binding epitope derived from the SKIP protein could be determined using a peptide array. A 36-residue region of SKIP centred on an eight-residue epitope suffices to bind PPIL1 in pull-down experiments. The crystal structure of PPIL1 in complex with the inhibitor cyclosporine A (CsA) was obtained at a resolution of 1.15 Å and exhibited two bound Cd2+ ions that enabled SAD phasing. PPIL1 residues that have previously been implicated in binding of SKIP are involved in the coordination of Cd2+ ions in the present crystal structure. Employing the present crystal structure, the determined minimal binding epitope and previously published NMR data [1], a molecular docking study was performed. In the docked model of the PPIL1·SKIP interaction, a proline residue of SKIP is buried in a hydrophobic pocket of PPIL1. This hydrophobic contact is encircled by several hydrogen bonds between the SKIP peptide and PPIL1. Conclusion We characterized a short, linear epitope of SKIP that is sufficient to bind the PPIL1 protein. Our data indicate that this SKIP peptide could function in recruiting PPIL1 into the core of the spliceosome. We present a molecular model for the binding mode of SKIP to PPIL1 which emphasizes the versatility of cyclophilin-type PPIases to engage in additional interactions with other proteins apart from active site contacts despite their limited surface area. PMID:20368803

  12. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus.

    PubMed

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  13. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

    PubMed Central

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

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

    PubMed Central

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

    2016-01-01

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

  15. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees.

    PubMed Central

    Goudsmit, J; Debouck, C; Meloen, R H; Smit, L; Bakker, M; Asher, D M; Wolff, A V; Gibbs, C J; Gajdusek, D C

    1988-01-01

    Chimpanzees are susceptible to infection by divergent strains of human immunodeficiency virus type 1 (HIV-1), none of which cause clinical or immunological abnormalities. Chimpanzees were inoculated with one of four strains of HIV-1: human T-lymphotropic virus (HTLV) type IIIB, lymphadenopathy virus (LAV) type 1, HTLV type IIIRF, or an isolate from the brain of a patient with acquired immunodeficiency syndrome. Within 6 months after inoculation with the closely related strains HTLV-IIIB or LAV-1, six chimpanzees developed serum antibodies to the C-terminal half (amino acids 288-467) of the HTLV-IIIB external envelope glycoprotein gp120. Sera from five of those chimpanzees had HTLV-IIIB cell-fusion-inhibiting antibody titers greater than or equal to 20 at that time, indicating that they neutralized the infecting strain of HIV-1 in vitro. No antibodies to the carboxyl terminus of HTLV-IIIB gp120 were observed in sera of chimpanzees inoculated with HTLV-IIIRF or with the brain-tissue strain, and those sera did not neutralize HTLV-IIIB. A rabbit immunized with the C-terminal portion of gp120 acquired neutralizing antibodies that bound to four domains of the HTLV-IIIB external envelope as analyzed by reactivity to 536 overlapping nonapeptides of gp120. One of these domains in the variable region V3, with the amino acid sequence IRIQRGPGRAFVTIG (amino acids 307-321), bound to all chimpanzee sera that neutralized HTLV-IIIB but not to the serum of the HTLV-IIIRF-inoculated chimpanzee that did not neutralize HTLV-IIIB. The HTLV-IIIRF sequence at the same location, ITKGPGRVIYA, was recognized by the serum of the HTLV-IIIRF-inoculated chimpanzee but not by any sera of the HTLV-IIIB-inoculated or LAV-1-inoculated chimpanzees. The HTLV-IIIB residues RIQR and AFV and the HTLV-IIIRF residues lysine and VIYA, flanking a highly conserved beta-turn (GPGR), appear to be critical for antibody binding and subsequent type-specific virus neutralization. This neutralization epitope

  16. Modulating the immunological properties of a linear B-cell epitope by insertion into permissive sites of the MalE protein.

    PubMed

    Martineau, P; Leclerc, C; Hofnung, M

    1996-12-01

    In a previous study, a set of positions in the MalE protein from Escherichia coli were identified, which tolerated short insertions or deletions without compromising the maltose binding activity of the protein. It is now shown that these sites accommodate an insert of 13 amino acids and are, therefore, permissive. Eleven sites were used, including eight permissive sites, to display a linear neutralization B-cell epitope of poliovirus (C3 epitope) at different positions on the surface of MalE. The affinity of a monoclonal neutralizing anti-poliovirus antibody (anti-C3 mAb) for the hybrid proteins varied from undetectable, to more than 1000 times higher than for the synthetic peptide. Therefore, some MalEC3 proteins mimic interactions of the viral epitope with the monoclonal antibody more efficiently than the free peptide. The results are interpreted in terms of the mobility of the insert and its flanking regions. It was further shown that some of the purified hybrid proteins are able to induce high titer anti-C3-peptide antibodies in mice. A strong correlation exists between the capacity of a MalEC3 protein to induce anti-C3-peptide antibodies and the antigenicity of the inserted peptide, measured with a polyclonal serum raised against the synthetic peptide. PMID:9171894

  17. Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

    PubMed Central

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

    2015-01-01

    Background The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis. Methodology/Principal Findings We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis. Conclusions/Significance The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions. PMID:25569432

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development. PMID:25258481

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

    PubMed Central

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

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development. PMID:25258481

  1. Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

    PubMed Central

    Kam, Yiu-Wing; Lum, Fok-Moon; Teo, Teck-Hui; Lee, Wendy W L; Simarmata, Diane; Harjanto, Sumitro; Chua, Chong-Long; Chan, Yoke-Fun; Wee, Jin-Kiat; Chow, Angela; Lin, Raymond T P; Leo, Yee-Sin; Le Grand, Roger; Sam, I-Ching; Tong, Joo-Chuan; Roques, Pierre; Wiesmüller, Karl-Heinz; Rénia, Laurent; Rötzschke, Olaf; Ng, Lisa F P

    2012-01-01

    Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally-acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope ‘E2EP3’. E2EP3 is located at the N-terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3-specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV-specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non-human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre-clinical basis for the design of effective vaccine against arthralgia-inducing CHIKV and other alphaviruses. PMID:22389221

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

    PubMed

    Hua, Rong-Hong; Liu, Li-Ke; Chen, Zhen-Shi; Li, Ye-Nan; Bu, Zhi-Gao

    2013-01-01

    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 (5)AIDITRK(11), (72)RDELNVL(78), (251)KSKHNRREGY(260), (269)DENGIVLD(276), and (341)DETTLVRS(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. PMID:23825668

  3. Conformational and Linear B-Cell Epitopes of Asp f 2, a Major Allergen of Aspergillus fumigatus, Bind Differently to Immunoglobulin E Antibody in the Sera of Allergic Bronchopulmonary Aspergillosis Patients

    PubMed Central

    Banerjee, Banani; Greenberger, Paul A.; Fink, Jordan N.; Kurup, Viswanath P.

    1999-01-01

    Asp f 2 is a major Aspergillus fumigatus allergen involved in allergic bronchopulmonary aspergillosis. Knowledge of the B-cell epitopes may contribute to the understanding of immunoregulation and immunodiagnosis. To elucidate the immunoglobulin E (IgE) binding epitopes in the linear sequence of Asp f 2, we synthesized decamer peptides spanning the whole molecule of Asp f 2 on derivatized cellulose membranes and evaluated IgE binding in ABPA patient and control sera. Peptides three to five amino acids long were synthesized based on amino acid sequences within the IgE binding regions and evaluated for the specificity of epitope antibody interactions. Nine IgE binding regions were recognized in this protein of 268 amino acid residues. Of the nine epitopes, seven (ATQRRQI, RKYFG, HWR, YTTRR, DHFAD, ALEAYA, and THEGGQ) are present in the hydrophilic regions of Asp f 2. Immunologic evaluation of the three recombinant fragments, Asp f 2A encompassing the N-terminal epitope region, Asp f 2B without N- and C-terminal regions of the protein, and Asp f 2C representing C-terminal epitopes, revealed that either the N- or C-terminal region of the protein is essential for the correct folding and conformation for IgE antibody binding. PMID:10225885

  4. Diagnostic Potential of Zinc Finger Protein-Specific Autoantibodies and Associated Linear B-Cell Epitopes in Colorectal Cancer

    PubMed Central

    O’Reilly, Julie-Ann; Fitzgerald, Jenny; Fitzgerald, Seán; Kenny, Dermot; Kay, Elaine W.; O’Kennedy, Richard; Kijanka, Gregor S.

    2015-01-01

    Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10–20% of colorectal cancer patients and in 0–5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer. PMID:25875936

  5. Mapping and modeling of a strain-specific epitope in the Norwalk virus capsid inner shell.

    PubMed

    Parra, Gabriel I; Sosnovtsev, Stanislav V; Abente, Eugenio J; Sandoval-Jaime, Carlos; Bok, Karin; Dolan, Michael A; Green, Kim Y

    2016-05-01

    Noroviruses are diverse positive-strand RNA viruses associated with acute gastroenteritis. Cross-reactive epitopes have been mapped primarily to conserved sequences in the capsid VP1 Shell (S) domain, and strain-specific epitopes to the highly variable Protruding (P) domain. In this work, we investigated a strain-specific linear epitope defined by MAb NV10 that was raised against prototype (Genogroup I.1) strain Norwalk virus (NV). Using peptide scanning and mutagenesis, the epitope was mapped to amino acids 21-32 (LVPEVNASDPLA) of the NV S domain, and its specificity was verified by epitope transfer and reactivity with a recombinant MAb NV10 single-chain variable fragment (scFv). Comparative structural modeling of the NV10 strain-specific and the broadly cross-reactive TV20 epitopes identified two internal non-overlapping sites in the NV shell, corresponding to variable and conserved amino acid sequences among strains, respectively. The S domain, like the P domain, contains strain-specific epitopes that contribute to the antigenic diversity among the noroviruses. PMID:26971245

  6. Linear stability of shock profiles for systems of conservation laws with semi-linear relaxation

    NASA Astrophysics Data System (ADS)

    Godillon, Pauline

    2001-01-01

    The Evans function theory, which has recently been applied to the study of linear stability of viscous shock profiles, is developed below for semi-linear relaxation. We study the linear stability of shock profiles in the Lax, undercompressive and overcompressive cases. The results we obtain are similar to those found for viscous approximations by Gardner and Zumbrun [Commun. Pure Appl. Math. 51 (7) (1998) 797].

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  8. High resolution numerical simulation of the linearized Euler equations in conservation law form

    NASA Technical Reports Server (NTRS)

    Sreenivas, Kidambi; Whitfield, David L.; Huff, Dennis L.

    1993-01-01

    A linearized Euler solver based on a high resolution numerical scheme is presented. The approach is to linearize the flux vector as opposed to carrying through the complete linearization analysis with the dependent variable vector written as a sum of the mean and the perturbed flow. This allows the linearized equations to be maintained in conservation law form. The linearized equations are used to compute unsteady flows in turbomachinery blade rows arising due to blade vibrations. Numerical solutions are compared to theoretical results (where available) and to numerical solutions of the nonlinear Euler equations.

  9. Immunogenecity of synthetic peptides representing linear B-cell epitopes of VapA of Rhodococcus equi.

    PubMed

    Taouji, Saïd; Nomura, Izumi; Giguère, Steeve; Tomomitsu, Seiji; Kakuda, Tsutomu; Ganne, Vincent; Takaï, Shinji

    2004-03-12

    Amino acid 65-78 of membrane protein VapA of the facultative intracellular Rhodococcus equi contained an immunodominant N-terminal B-cell epitope (N15Y peptide). Safety and immunogenecity of a synthetic peptide consisting of the amino acid 65-78 of VapA (peptide N15Y) were evaluated first in mice and in healthy adult horses. A single dose of a peptide-VapA vaccine induced and only in presence of adjuvant, specific IgG antibodies in sera of mice. After challenge with virulent R. equi 3 weeks after immunization, tissue clearance was more delayed in immunized mice than in control mice. An antibody-mediated response (restricted to IgG1 and IgG2b subclasses) predominated in vaccinated mice sera and no specific lymphocytes proliferation was observed. Next, a total of 15 mares were given systemic inoculation of N15Y peptide with IMS3012 ( n = 4 ) or IMS2211 ( n = 4 ) or ISA35 ( n = 4 ) or placebo ( n = 2). Serological responses to the peptide vaccine were found in all but not in placebo group. A significant increase of IgGb subclass in sera of vaccinated mare with N15Y peptide in presence of IMS3012 was observed in comparison to IMS2211 or ISA35 or control group. Moreover, INF-gamma, IL-2 and IL-10 mRNA expression increased more significantly in peripheral blood lymphocytes of IMS3012 group than in IMS2211 or ISA35 group. Interestingly, a significant decrease of IL-4 mRNA expression (undetectable level) was observed with all adjuvants. These results support the use of peptide N15Y in presence of IMS3012 adjuvant in future studies of protection of foals against R. equi. PMID:15003638

  10. Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus.

    PubMed

    Odongo, David; Kamau, Lucy; Skilton, Robert; Mwaura, Stephen; Nitsch, Cordula; Musoke, Anthony; Taracha, Evans; Daubenberger, Claudia; Bishop, Richard

    2007-01-26

    Vaccines based on recombinant Bm86 gut antigen from Boophilus microplus are a useful component of integrated control strategies against B. microplus infestations of cattle. The capacity of such vaccines to control heterologous infestations by two African tick species was investigated. The mean weight of engorged female ticks and mean egg mass per tick were significantly reduced in B. decoloratus infestations, but there was no effect of the vaccine against adult Rhipicephalus appendiculatus. We cloned, sequenced and expressed two Bm86 homologues (Bd86) from B. decoloratus. Amino acid sequence identity between Bd86 homologues (Bd86-1 and Bd86-2) and Bm86 was 86% and 85%, respectively, compared to 93% identity between the variants. Native Bd86 protein in B. decoloratus tick mid-gut sections and recombinant Bd86-1 reacted strongly with sera from TickGARD vaccinated cattle. TickGARD can therefore protect against a heterologous tick species with multiple antigen sequences. Epitope mapping using sera from TickGARD-vaccinated cattle identified two linear peptides conserved between the Bd86 homologues and Bm86. These epitopes represent candidate synthetic peptide vaccines for control of Boophilus spp. and the pathogens transmitted by these tick vectors. PMID:17070625

  11. Epitopes for Broad and Potent Neutralizing Antibody Responses during Chronic Infection with Human Immunodeficiency Virus Type 1

    PubMed Central

    Nandi, Avishek; Lavine, Christine L.; Wang, Pengcheng; Lipchina, Inna; Goepfert, Paul A.; Shaw, George M.; Tomaras, Georgia D.; Montefiori, David C.; Haynes, Barton F.; Easterbrook, Philippa; Robinson, James E.; Sodroski, Joseph G.; Yang, Xinzhen

    2009-01-01

    Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved non-linear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection. PMID:19922969

  12. Structural basis for epitope sharing between group 1 allergens of cedar pollen.

    PubMed

    Midoro-Horiuti, Terumi; Schein, Catherine H; Mathura, Venkatarajan; Braun, Werner; Czerwinski, Edmund W; Togawa, Akihisa; Kondo, Yasuto; Oka, Tetsuo; Watanabe, Masanao; Goldblum, Randall M

    2006-02-01

    The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development. PMID:15975657

  13. Structural basis for epitope sharing between group 1 allergens of cedar pollen

    PubMed Central

    Midoro-Horiuti, Terumi; Schein, Catherine H.; Mathura, Venkatarajan; Braun, Werner; Czerwinski, Edmund W.; Togawa, Akihisa; Kondo, Yasuto; Oka, Tetsuo; Watanabe, Masanao; Goldblum, Randall M.

    2008-01-01

    The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved β-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development. PMID:15975657

  14. Bianchi-Baecklund transformations, conservation laws, and linearization of various field theories

    SciTech Connect

    Chau Wang, L L

    1980-01-01

    The discussion includes: the Sine-Gordon equation, parametric Bianchi-Baecklund transformations and the derivation of local conservation laws; chiral fields, parametric Bianchi-Baecklund transformations, local and non-local conservation laws, and linearization; super chiral fields, a parallel development similar to the chiral field; and self-dual Yang-Mills fields in 4-dimensional Euclidean space; loop-cpace chiral equations, parallel development but with subtlety. (GHT)

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  16. Identification of a novel B-cell epitope specific for avian leukosis virus subgroup J gp85 protein.

    PubMed

    Li, Xiaofei; Zhu, Haibo; Wang, Qi; Sun, Jiashan; Gao, Yanni; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2015-04-01

    Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused severe economic losses in China. Gp85 protein is the main envelope protein and the most variable structural protein of ALV-J. It is also involved in virus neutralization. In this study, a specific monoclonal antibody, 4A3, was produced against the ALV-J gp85 protein. Immunofluorescence assays showed that 4A3 could react with different strains of ALV-J, including the British prototype isolate HPRS103, the American strains, an early Chinese broiler isolate, and layer isolates. A linear epitope on the gp85 protein was identified using a series of partially overlapping fragments spanning the gp85-encoding gene and subjecting them to western blot analysis. The results indicated that (134)AEAELRDFI(142) was the minimal linear epitope that could be recognized by mAb 4A3. Enzyme-linked immunosorbent assay (ELISA) revealed that chicken anti-ALV-J sera and mouse anti-ALV-J gp85 sera could also recognize the minimal linear epitope. Alignment analysis of amino acid sequences indicated that the epitope was highly conserved among 34 ALV-J strains. Furthermore, the epitope was not conserved among subgroup A and B of avian leukosis virus (ALV). Taken together, the mAb and the identified epitope may provide valuable tools for the development of new diagnostic methods for ALV-J. PMID:25655260

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

    PubMed

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

    2015-01-01

    We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB(*)) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB(*) adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections. PMID:25767809

  18. Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1.

    PubMed

    HaiXiu, Wang; EnCheng, Sun; QingYuan, Xu; Tao, Yang; Qin, Zhang; YuFei, Feng; JunPing, Li; Shuang, Lv; Liang, Sun; Jing, Sun; DongLai, Wu

    2015-02-01

    The bluetongue virus (BTV) NS1 protein is one of the major proteins synthesized during BTV infection and is responsible for the generation of virus-specific tubules. Although some functional and structural studies on the BTV NS1 protein have been reported, there have been no reports describing the linear B-cell epitopes recognized by humoral immune responses published to date. In this study, 25 BTV12 NS1-reactive monoclonal antibodies (MAbs) and polyclonal antisera (polyclonal antibodies, PAbs) were generated and analyzed. We identified 14 linear NS1 epitopes recognized by the PAbs and MAbs using NS1-derived peptides in an enzyme-linked immunosorbent assay. Moreover, we predicted 23 linear B-cell epitopes using the ABCpred online server which employs an artificial neural network. Analysis of the predicted and identified epitopes of NS1 demonstrated the feasibility of B-cell epitope prediction. Sequence alignments indicated that the epitopes recognized by MAbs are highly conserved among BTV serotypes, but not among the other members of the genus Orbivirus, such as the African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Chuzan disease virus (CV). Importantly, we identified specific MAbs that recognized all BTV serotypes tested as well as MAbs that recognized only BTV12, suggesting that these NS1-specific MAbs could serve as a basis for BTV diagnostic approaches. The generation and identification of NS1 protein epitopes will provide the foundation for further studies about the function and structure of NS1 and novel epitope-based vaccines. PMID:25343975

  19. Linearized stability of extreme shock profiles for systems of conservation laws with viscosity

    NASA Astrophysics Data System (ADS)

    Pego, R. L.

    1982-12-01

    For a genuinely nonlinear hyperbolic system of conservation laws with added artificial viscosity, we prove that traveling wave profiles for small amplitude extreme shocks (the slowest and fastest) are linearly stable to perturbations initial data chosen from certain spaces with weighted norm; i.e., we show that the spectrum of the linearized equation lies strictly in the left half plane, except for a simple eigenvalue at the origin (due to phase translations of the profile). The weight is used in components transverse to the profile, where, for an extreme shock, the linearized equation is dominated by unidirectional convection.

  20. 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.

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  3. Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases

    PubMed Central

    Li, Sijin; Sun, Jing; Teng, Yumei; Wu, Meini; Li, Jianfan; Li, Yanhan; Hu, Ningzhu; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases. PMID:26641892

  4. Dynamic scaling behaviors of linear fractal Langevin-type equation driven by nonconserved and conserved noise

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Xun, Zhi-Peng; Wu, Ling; Chen, Yi-Li; Xia, Hui; Hao, Da-Peng; Tang, Gang

    2016-06-01

    In order to study the effects of the microscopic details of fractal substrates on the scaling behavior of the growth model, a generalized linear fractal Langevin-type equation, ∂h / ∂t =(- 1) m + 1 ν∇ mzrw h (zrw is the dynamic exponent of random walk on substrates), driven by nonconserved and conserved noise is proposed and investigated theoretically employing scaling analysis. Corresponding dynamic scaling exponents are obtained.

  5. On the linearized stability of viscous shock profiles for systems of conservation laws

    NASA Astrophysics Data System (ADS)

    Xin, Zhouping

    This paper is concerned with the linearized stability of traveling wave solutions for systems of viscous hyperbolic conservation laws. The main purpose is to show that for a given traveling wave with shock profile from any characteristic family, there exists an appropriate weighted norm space such that the traveling wave is exponentially stable in this space. As a consequence, if the initial disturbance has average zero and decays exponentially fast as ¦x¦ → ∞, then the corresponding solution of the linearized equation decays to zero exponentially fast in t on any compact interval in x. The proof is given by applying an elementary weighted characteristic energy method to the integrated linearized system, based on the underlying wave structure.

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

    PubMed

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

    2016-02-01

    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

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

    PubMed Central

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

    2016-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

    PubMed

    Jelsma, H; Loeffen, W L A; van Beuningen, A; van Rijn, P A

    2013-09-27

    Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed. PMID:23838147

  18. Prediction of Antibody Epitopes.

    PubMed

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin. Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody epitopes from the sequence and/or the three-dimensional structure of a target protein. PMID:26424260

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

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

  1. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

    PubMed

    Foo, D G W; Ang, R X; Alonso, S; Chow, V T K; Quak, S H; Poh, C L

    2008-03-01

    A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6. PMID:18076666

  2. Counter-propagating dual-trap optical tweezers based on linear momentum conservation

    SciTech Connect

    Ribezzi-Crivellari, M.; Huguet, J. M.; Ritort, F.

    2013-04-15

    We present a dual-trap optical tweezers setup which directly measures forces using linear momentum conservation. The setup uses a counter-propagating geometry, which allows momentum measurement on each beam separately. The experimental advantages of this setup include low drift due to all-optical manipulation, and a robust calibration (independent of the features of the trapped object or buffer medium) due to the force measurement method. Although this design does not attain the high-resolution of some co-propagating setups, we show that it can be used to perform different single molecule measurements: fluctuation-based molecular stiffness characterization at different forces and hopping experiments on molecular hairpins. Remarkably, in our setup it is possible to manipulate very short tethers (such as molecular hairpins with short handles) down to the limit where beads are almost in contact. The setup is used to illustrate a novel method for measuring the stiffness of optical traps and tethers on the basis of equilibrium force fluctuations, i.e., without the need of measuring the force vs molecular extension curve. This method is of general interest for dual trap optical tweezers setups and can be extended to setups which do not directly measure forces.

  3. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

    PubMed

    Lai, Chih-Yun; Tsai, Wen-Yang; Lin, Su-Ru; Kao, Chuan-Liang; Hu, Hsien-Ping; King, Chwan-Chuen; Wu, Han-Chung; Chang, Gwong-Jen; Wang, Wei-Kung

    2008-07-01

    The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well. PMID:18448542

  4. Ab and T cell epitopes of influenza A virus, knowledge and opportunities

    PubMed Central

    Bui, Huynh-Hoa; Peters, Bjoern; Assarsson, Erika; Mbawuike, Innocent; Sette, Alessandro

    2007-01-01

    The Immune Epitope Database and Analysis Resources (IEDB) (www.immuneepitope.org) was recently developed to capture epitope related data. IEDB also hosts various bioinformatics tools that can be used to identify novel epitopes as well as to analyze and visualize existing epitope data. Herein, a comprehensive analysis was undertaken (i) to compile and inventory existing knowledge regarding influenza A epitopes and (ii) to determine possible cross-reactivities of identified epitopes among avian H5N1 and human influenza strains. At present, IEDB contains >600 different epitopes derived from 58 different strains and 10 influenza A proteins. By using the IEDB analysis resources, conservancy analyses were performed, and several conserved and possibly cross-reactive epitopes were identified. Significant gaps in the current knowledge were also revealed, including paucity of Ab epitopes in comparison with T cell epitopes, limited number of epitopes reported for avian influenza strains/subtypes, and limited number of epitopes reported from proteins other than hemagglutinin and nucleoprotein. This analysis provides a resource for researchers to access existing influenza epitope data. At the same time, the analysis illustrates gaps in our collective knowledge that should inspire directions for further study of immunity against the influenza A virus. PMID:17200302

  5. Presence of potential allergy-related linear epitopes in novel proteins from conventional crops and the implication for the safety assessment of these crops with respect to the current testing of genetically modified crops.

    PubMed

    Kleter, Gijs A; Peijnenburg, Ad A C M

    2003-09-01

    Mitochondria of cytoplasmic male sterile crop plants contain novel, chimeric open reading frames. In addition, a number of crops carry endogenous double-stranded ribonucleic acid (dsRNA). In this study, the novel proteins encoded by these genetic components were screened for the presence of potential binding sites (epitopes) of allergy-associated IgE antibodies, as was previously done with transgenic proteins from genetically modified crops. The procedure entails the identification of stretches of at least six contiguous amino acids that are shared by novel proteins and known allergenic proteins. These stretches are further checked for potential linear IgE-binding epitopes. Of the 16 novel protein sequences screened in this study, nine contained stretches of six or seven amino acids that were also present in allergenic proteins. Four cases of similarity are of special interest, given the predicted antigenicity of the identical stretch within the allergenic and novel protein, the IgE-binding by a peptide containing an identical stretch reported in literature, or the multiple incidence of identical stretches of the same allergen within a novel protein. These selected stretches are present in novel proteins derived from oilseed rape and radish (ORF138), rice (dsRNA), and fava bean (dsRNA), and warrant further clinical testing. The frequency of positive outcomes and the sizes of the identical stretches were comparable to those previously found for transgenic proteins in genetically modified crops. It is discussed whether novel proteins from conventional crops should be subject to an assessment of potential allergenicity, a procedure which is currently mandatory for transgenic proteins from genetically modified crops. PMID:17166136

  6. The numerical simulation of non-linear non-radial stellar pulsations: a conservative formulation of gravity

    NASA Astrophysics Data System (ADS)

    Glatzel, W.; Chernigovski, S.

    2016-04-01

    The simulation of finite amplitude stellar pulsations and the theoretical determination of the final fate of violently unstable stellar models require the complete consideration of the mechanics and thermodynamics of a star. In particular, non-linear effects need to be taken into account. Numerical methods for the study of non-linear pulsations are available so far only for spherically symmetric radial pulsations. This paper is meant as a first step towards a numerical scheme which allows for the representation of non-radial non-linear stellar pulsations. A characteristic of stellar pulsations are dominant gravitational and thermal energies exceeding the kinetic energy by several orders of magnitude. As a consequence, numerical simulations of stellar pulsations require an extremely high accuracy, which can be met only by sophisticated intrinsically strictly conservative numerical schemes. Whether gravity can be represented numerically in a fully conservative way is the subject of this study. A fully conservative discrete form of gravity is derived explicitly and proven to satisfy all common conservation laws intrinsically.

  7. Properties of finite difference models of non-linear conservative oscillators

    NASA Technical Reports Server (NTRS)

    Mickens, R. E.

    1988-01-01

    Finite-difference (FD) approaches to the numerical solution of the differential equations describing the motion of a nonlinear conservative oscillator are investigated analytically. A generalized formulation of the Duffing and modified Duffing equations is derived and analyzed using several FD techniques, and it is concluded that, although it is always possible to contstruct FD models of conservative oscillators which are themselves conservative, caution is required to avoid numerical solutions which do not accurately reflect the properties of the original equation.

  8. The Conservation/Solution Element (STE) Method for Linear Potential Flow Problems

    NASA Technical Reports Server (NTRS)

    Adeyeye, John O.; Attia, Naguib F.; Jackson, Joy; Hunter, Timothy

    1996-01-01

    The potential equation is discretized on rectangular domains using the Conservation/Solution Element Method (STE) approach. Computational examples with a discussion of numerical experience gained are given.

  9. BABESIA BOVIS MEROZOITE SURFACE PROTEIN-2C (MSA-2C) CONTAINS HIGHLY IMMUNOGENIC, CONSERVED B-CELL EPITOPES THAT ELICIT NEUTRALIZATION-SENSITIVE ANTIBODIES IN CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The search for vaccine candidates against bovine babesiosis caused by Babesia bovis is greatly focused on the identification of merozoite surface-exposed antigens that are widely conserved, functionally relevant and immunodominant in cattle protected against B. bovis infections. We have recently id...

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

    PubMed

    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

  11. Nonlinear (time domain) and linearized (time and frequency domain) solutions to the compressible Euler equations in conservation law form

    NASA Technical Reports Server (NTRS)

    Sreenivas, Kidambi; Whitfield, David L.

    1995-01-01

    Two linearized solvers (time and frequency domain) based on a high resolution numerical scheme are presented. The basic approach is to linearize the flux vector by expressing it as a sum of a mean and a perturbation. This allows the governing equations to be maintained in conservation law form. A key difference between the time and frequency domain computations is that the frequency domain computations require only one grid block irrespective of the interblade phase angle for which the flow is being computed. As a result of this and due to the fact that the governing equations for this case are steady, frequency domain computations are substantially faster than the corresponding time domain computations. The linearized equations are used to compute flows in turbomachinery blade rows (cascades) arising due to blade vibrations. Numerical solutions are compared to linear theory (where available) and to numerical solutions of the nonlinear Euler equations.

  12. Conservation.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

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

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

    PubMed

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

    2016-02-01

    Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA. PMID:26612210

  14. Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts.

    PubMed

    Saeed, Hanaa; Gagnon, Christine; Cober, Elroy; Gleddie, Steve

    2016-02-01

    Soybean consumption is increasing in many Western diets; however, recent reviews suggest that the prevalence of soy allergy can be as high as 0.5% for the general population and up to 13% for children. The immunoglobulin-E (IgE) binding of sera from six soy-sensitive adult human subjects to soybean proteins separated by 2D gel electrophoresis was studied. Synthetic peptide sets spanning the mature glycinin subunit A2 and A3 primary sequences were used to map the IgE-binding regions. Putative epitopes identified in this study were also localized on glycinin hexamer models using bioinformatics software. We identified linear IgE-binding epitopes of the major storage protein Gly m 6 by screening individual soy-sensitive patient sera. These epitopes were then further analysed by 3D in silico model localization and compared to other plant storage protein epitopes. Web-based software applications were also used to study the ability to accurately predict epitopes with mixed results. A total of nine putative IgE-binding epitopes were identified in the glycinin A3 (A3.1-A3.3) and A2 (A2.1-A2.6) subunits. Most patients' sera IgE bound to only one or two epitopes, except for one patient's serum which bound to four different A2 epitopes. Two epitopes (A3.2 and A2.4) overlapped with a previously identified epitope hot spot of 11S globulins from other plant species. Most epitopes were predicted to be exposed on the surface of the 3D model of the glycinin hexamer. Amino acid sequence alignments of soybean acidic glycinins and other plant globulins revealed one dominant epitope hot spot among the four reported hot spots. This study may be helpful for future development of soy allergy immunotherapy and diagnosis. PMID:26766775

  15. Non-linear energy conservation theorem in the framework of special relativity

    NASA Astrophysics Data System (ADS)

    Pérez Teruel, Ginés R.

    2015-07-01

    In this work we revisit the study of the gravitational interaction in the context of the special theory of relativity. It is found that, as long as the equivalence principle is respected, a relativistic nonlinear energy conservation theorem arises in a natural way. We interpret that this nonlinear conservation law stresses the nonlinear character of the gravitational interaction. The theorem reproduces the energy conservation theorem of Newtonian mechanics in the corresponding low energy limit, but also allows to derive some standard results of post-Newtonian gravity, such as the formula of the gravitational redshift. Guided by this conservation law, we develop a Lagrangian formalism for a particle in a gravitational field. We realize that the Lagrangian can be written in an explicit covariant fashion, and turns out to be the geodesic Lagrangian of a curved Lorentzian manifold. Therefore, any attempt to describe gravity within the special theory, leads outside their own domains towards a curved space-time. Thus, the pedagogical content of the paper may be useful as a starting point to discuss the problem of gravitation in the context of the special theory, as a preliminary step before introducing general relativity.

  16. HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

    PubMed

    Wang, Yuge; Kapoor, Pratibha; Parks, Robert; Silva-Sanchez, Aaron; Alam, S Munir; Verkoczy, Laurent; Liao, Hua-Xin; Zhuang, Yingxin; Burrows, Peter; Levinson, Michael; Elgavish, Ada; Cui, Xiangqin; Haynes, Barton F; Schroeder, Harry

    2016-02-01

    Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization. PMID:26687685

  17. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    SciTech Connect

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-05-05

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of /sup 125/I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10/sup -8/ to 1 x 10/sup -6/ M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca/sup 2 +/, Mg/sup 2 +/, or Mn/sup 2 +/ partially inhibited binding of /sup 125/I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved ..gamma..-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins.

  18. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  1. Characterization of a key neutralizing epitope on pertussis toxin recognized by the monoclonal antibody 1B7

    PubMed Central

    Sutherland, Jamie N; Maynard, Jennifer A

    2009-01-01

    Despite over five decades of research and vaccination, infection by Bordetella pertussis remains a serious disease with no specific treatments or validated correlates of protective immunity. Of the numerous monoclonal antibodies binding pertussis toxin (PTx) that have been produced and characterized, the murine IgG2a monoclonal antibody 1B7 is uniquely neutralizing in all in vitro assays and in vivo murine models of infection. 1B7 binds an epitope on the enzymatically active S1-subunit of PTx (PTx-S1) with some linear elements but previous work with S1 scanning peptides, phage displayed peptide libraries, and S1 truncation/deletion variants were unable to more precisely define the epitope. Using computational docking algorithms, alanine scanning mutagenesis, and surface plasmon resonance, we characterize the epitope bound by 1B7 on PTx-S1 in molecular detail and define energetically important interactions between residues at the interface. Six residues on PTx-S1 and six residues on 1B7 were identified which, when altered to alanine, resulted in variants with significantly reduced affinity for the native partner. Using this information, a model of the 1B7-S1 interaction was developed, indicating a predominantly conformational epitope located on the base of S1 near S4. The location of this epitope is consistent with previous data and is shown to be conserved across several naturally occurring strain variants including PTx-S1A, B (Tohama-I), D, and E (18-323) in addition to the catalytically inactive 9K/129G variant. This highly neutralizing but poorly immunogenic epitope may represent an important target for next generation vaccine development, identification of immune correlates and passive immunization strategies in pertussis. PMID:19899804

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

    PubMed

    Sun, Encheng; Zhao, Jing; Liu, Nihong; Yang, Tao; Xu, Qingyuan; Qin, Yongli; Bu, Zhigao; Yang, Yinhui; Lunt, Ross A; Wang, Linfa; Wu, Donglai

    2012-01-01

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

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

    PubMed Central

    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. PMID:24964950

  4. Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

    PubMed Central

    2010-01-01

    Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong

  5. A new class of non-linear stochastic population models with mass conservation.

    PubMed

    Kooijman, S A L M; Grasman, J; Kooi, B W

    2007-12-01

    We study the effects of random feeding, growing and dying in a closed nutrient-limited producer/consumer system, in which nutrient is fully conserved, not only in the mean, but, most importantly, also across random events. More specifically, we relate these random effects to the closest deterministic models, and evaluate the importance of the various times scales that are involved. These stochastic models differ from deterministic ones not only in stochasticity, but they also have more details that involve shorter times scales. We tried to separate the effects of more detail from that of stochasticity. The producers have (nutrient) reserve and (body) structure, and so a variable chemical composition. The consumers have only structure, so a constant chemical composition. The conversion efficiency from producer to consumer, therefore, varies. The consumers use reserve and structure of the producers as complementary compounds, following the rules of Dynamic Energy Budget theory. Consumers die at constant specific rate and decompose instantaneously. Stochasticity is incorporated in the behaviour of the consumers, where the switches to handling and searching, as well as dying are Poissonian point events. We show that the stochastic model has one parameter more than the deterministic formulation without time scale separation for conversions between searching and handling consumers, which itself has one parameter more than the deterministic formulation with time scale separation for these conversions. These extra parameters are the contributions of a single individual producer and consumer to their densities, and the ratio of the two, respectively. The tendency to oscillate increases with the number of parameters. The focus bifurcation point has more relevance for the asymptotic behaviour of the stochastic model than the Hopf bifurcation point, since a randomly perturbed damped oscillation exhibits a behaviour similar to that of the stochastic limit cycle particularly

  6. A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination.

    PubMed

    Nadin-Davis, S A; Sheen, M; Abdel-Malik, M; Elmgren, L; Armstrong, J; Wandeler, A I

    2000-04-01

    A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination. PMID:10747114

  7. Structure of allergens and structure based epitope predictions☆

    PubMed Central

    Dall’Antonia, Fabio; Pavkov-Keller, Tea; Zangger, Klaus; Keller, Walter

    2014-01-01

    The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction. PMID:23891546

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

    PubMed

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

    2016-04-01

    Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings. PMID:26970211

  9. Structure-Function Analysis of the Epitope for 4E10, a Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody†

    PubMed Central

    Brunel, Florence M.; Zwick, Michael B.; Cardoso, Rosa M. F.; Nelson, Josh D.; Wilson, Ian A.; Burton, Dennis R.; Dawson, Philip E.

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the Kd values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH2 could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope. PMID:16439525

  10. Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

    PubMed Central

    Fairlie, W D; Stanton, P G; Hearn, M T

    1995-01-01

    The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites. Images Figure 2 PMID:7538754

  11. Linear B-cell epitopes in BthTX-1, BthTX-II and BthA-1, phospholipase A₂'s from Bothrops jararacussu snake venom, recognized by therapeutically neutralizing commercial horse antivenom.

    PubMed

    De-Simone, Salvatore G; Napoleão-Pego, Paloma; Teixeira-Pinto, Luiz A L; Santos, Jonathas D L; De-Simone, Thatiane S; Melgarejo, Anibal R; Aguiar, Aniesse S; Marchi-Salvador, Daniela P

    2013-09-01

    The benefits from treatment with antivenom sera are indubitable. However, the mechanism for toxin neutralization has not been completely elucidated. A mixture of anti-bothropic and anti-crotalic horse antivenom has been reported to be more effective in neutralizing the effects of Bothrops jararacussu snake venom than anti-bothropic antivenom alone. This study determined which regions in the three PLA₂s from B. jararacussu snake venom are bound by antibodies in tetravalent anti-bothropic and monovalent anti-crotalic commercial horse antivenom. Mapping experiments of BthTX-I, BthTX-II and BthA-I using two small libraries of 69 peptides each revealed six major IgG-binding epitopes that were recognized by both anti-bothropic and anti-crotalic horse antivenom. Two epitopes in BthTX-I were only recognized by the anti-bothropic horse antivenom, while anti-crotalic horse antivenom recognized four unique epitopes across the three PLA₂s. Our studies suggest that the harmful activities of the PLA₂s present in the venom of B. jararacussu are neutralized by the combinatorial treatment with both antivenom sera through their complementary binding sites, which provides a wide coverage on the PLA₂s. This is the first peptide microarray of PLA₂s from B. jararacussu snake venom to survey the performance of commercial horse antiophidic antivenom. Regions recognized by the protective antivenom sera are prime candidates for improved venom cocktails or a chimeric protein encoding the multiple epitopes to immunize animals as well as for designing future synthetic vaccines. PMID:23792452

  12. Relationship of the human immunodeficiency virus type 1 gp120 third variable loop to a component of the CD4 binding site in the fourth conserved region.

    PubMed Central

    Wyatt, R; Thali, M; Tilley, S; Pinter, A; Posner, M; Ho, D; Robinson, J; Sodroski, J

    1992-01-01

    Neutralizing antibodies that recognize the human immunodeficiency virus gp120 exterior envelope glycoprotein and are directed against either the third variable (V3) loop or conserved, discontinuous epitopes overlapping the CD4 binding region have been described. Here we report several observations that suggest a structural relationship between the V3 loop and amino acids in the fourth conserved (C4) gp120 region that constitute part of the CD4 binding site and the conserved neutralization epitopes. Treatment of the gp120 glycoprotein with ionic detergents resulted in a V3 loop-dependent masking of both linear C4 epitopes and discontinuous neutralization epitopes overlapping the CD4 binding site. Increased recognition of the native gp120 glycoprotein by an anti-V3 loop monoclonal antibody, 9284, resulted from from single amino acid changes either in the base of the V3 loop or in the gp120 C4 region. These amino acid changes also resulted in increased exposure of conserved epitopes overlapping the CD4 binding region. The replication-competent subset of these mutants exhibited increased sensitivity to neutralization by antibody 9284 and anti-CD4 binding site antibodies. The implied relationship of the V3 loop, which mediates post-receptor binding steps in virus entry, and components of the CD4 binding region may be important for the interaction of these functional gp120 domains and for the observed cooperativity of neutralizing antibodies directed against these regions. Images PMID:1279195

  13. Hepatitis C virus mutation affects proteasomal epitope processing

    PubMed Central

    Seifert, Ulrike; Liermann, Heike; Racanelli, Vito; Halenius, Anne; Wiese, Manfred; Wedemeyer, Heiner; Ruppert, Thomas; Rispeter, Kay; Henklein, Peter; Sijts, Alice; Hengel, Hartmut; Kloetzel, Peter-M.; Rehermann, Barbara

    2004-01-01

    The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2–restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2–positive and in 11/24 (46%) HLA-A2–negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2–restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2–transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-γ;–producing and fewer tetramer+ cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8+ T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients. PMID:15254592

  14. Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes - relevance for vaccine design.

    PubMed

    Denisova, Galina F; Denisov, Dimitri A; Bramson, Jonathan L

    2010-01-01

    To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific "signs" and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific "signs" and assigned to specific epitopes. We are currently using computational methods to define epitope "signs" without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera. PMID:21067548

  15. Number-conserving linear-response study of low-velocity ion stopping in a collisional magnetized classical plasma

    SciTech Connect

    Nersisyan, Hrachya B.; Deutsch, Claude; Das, Amal K.

    2011-03-15

    The results of a theoretical investigation of the low-velocity stopping power of ions in a magnetized collisional and classical plasma are reported. The stopping power for an ion is calculated through the linear-response (LR) theory. The collisions, which lead to a damping of the excitations in the plasma, are taken into account through a number-conserving relaxation time approximation in the LR function. In order to highlight the effects of collisions and magnetic field, we present a comparison of our analytical and numerical results obtained for nonzero damping or magnetic field with those for vanishing damping or magnetic field. It is shown that the collisions remove the anomalous friction obtained previously [Nersisyan et al., Phys. Rev. E 61, 7022 (2000)] for the collisionless magnetized plasmas at low ion velocities. One of the major objectives of this paper is to compare and to contrast our theoretical results with those obtained through a diffusion coefficient formulation based on the Dufty-Berkovsky relation evaluated for a magnetized one-component plasma modeled with target ions and electrons.

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

    PubMed

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

    2010-05-01

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

  17. Back to the future: covalent epitope-based HIV vaccine development

    PubMed Central

    Paul, Sudhir; Planque, Stephanie; Nishiyama, Yasuhiro; Escobar, Miguel; Hanson, Carl

    2010-01-01

    Traditional HIV vaccine approaches have proved ineffective because the immunodominant viral epitopes are mutable and the conserved epitopes necessary for infection are not sufficiently immunogenic. The CD4 binding site expressed by the HIV envelope protein of glycoprotein 120 is essential for viral entry into host cells. In this article, we review the B-cell superantigenic character of the CD4 binding site as the cause of its poor immunogenicity. We summarize evidence supporting development of covalent immunization as the first vaccine strategy with the potential to induce an antibody response to a conserved HIV epitope that neutralizes genetically divergent HIV strains. PMID:20822346

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

    PubMed

    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

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

    PubMed Central

    Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

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

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

    PubMed

    Li, Machao; Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

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

  1. Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes.

    PubMed

    Barre, Annick; Borges, Jean-Philippe; Culerrier, Raphaël; Rougé, Pierre

    2005-09-15

    Three-dimensional models built for the peanut Ara h 2 allergen and other structurally-related 2S albumin allergens of dietary nuts exhibited an overall three-dimensional fold stabilized by disulphide bridges well conserved among all the members of the 2S albumin superfamily. Conformational analysis of the linear IgE-binding epitopes mapped on the molecular surface of Ara h 2 showed no structural homology with the corresponding regions of the walnut Jug r 1, the pecan nut Car i 1 or the Brazil nut Ber e 1 allergens. The absence of epitopic community does not support the allergenic cross-reactivity observed between peanut and walnut or Brazil nut, which presumably depends on other ubiquitous seed storage protein allergens, namely the vicilins. However, the major IgE-binding epitope identified on the molecular surface of the walnut Jug r 1 allergen shared a pronounced structural homology with the corresponding region of the pecan nut Car i 1 allergen. With the exception of peanut, 2S albumins could thus account for the IgE-binding cross-reactivity observed between some other dietary nuts, e.g. walnut and pecan nut. PMID:15899521

  2. Allergen structures and epitopes.

    PubMed

    Meno, K H

    2011-07-01

    Human type 1 hypersensitivity diseases such as allergic rhinoconjunctivitis are characterized by allergen-specific IgE antibodies produced in allergic individuals after allergen exposure. IgE antibodies bound to receptors on the surface of effector cells trigger an allergic response by interacting with three-dimensional (conformational) epitopes on the allergen surface. Crystal structures are available for complexes of antibody specifically bound to five allergens, from birch pollen, bee venom, cockroach, cow's milk and timothy grass pollen. The details of the antibody-allergen interaction extending all the way to atomic resolution are available from such complexes. In vitro investigations using recombinant monoclonal antibodies and human basophils show that binding affinity is a key to triggering the allergic response. Continued molecular characterization of antibody-allergen interactions is paving the way for the use of recombinant allergens in allergen-specific diagnosis and immunotherapy. PMID:21668845

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

    PubMed Central

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

    2015-01-01

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

  4. A novel multi-epitope peptide vaccine against cancer: an in silico approach.

    PubMed

    Nezafat, Navid; Ghasemi, Younes; Javadi, Gholamreza; Khoshnoud, Mohammad Javad; Omidinia, Eskandar

    2014-05-21

    Cancer immunotherapy has an outstanding position in cancer prevention and treatment. In this kind of therapy, the immune system is activated to eliminate cancerous cells. Multi-epitope peptide cancer vaccines are manifesting as the next generation of cancer immunotherapy. In the present study, we have implemented various strategies to design an efficient multi-epitope vaccine. CD8+ cytolytic T lymphocytes (CTLs) epitopes, which have a pivotal role in cellular immune responses, helper epitopes and adjuvant, are three crucial components of peptide vaccine. CTL epitopes were determined from two high immunogenic protein Wilms tumor-1 (WT1) and human papillomavirus (HPV) E7 by various servers, which apply different algorithms. CTL epitopes were linked together by AAY and HEYGAEALERAG motifs to enhance epitope presentation. Pan HLA DR-binding epitope (PADRE) peptide sequence and helper epitopes, which have defined from Tetanus toxin fragment C (TTFrC) by various servers, were used to induce CD4+ helper T lymphocytes (HTLs) responses. Additionally, helper epitopes were conjugated together via GPGPG motifs that stimulate HTL immunity. Heparin-Binding Hemagglutinin (HBHA), a novel TLR4 agonist was employed as an adjuvant to polarize CD4+ T cells toward T-helper 1 to induce strong CTL responses. Moreover, the EAAAK linker was introduced to N and C terminals of HBHA for efficient separation. 3D model of protein was generated and predicted B cell epitopes were determined from the surface of built structure. Our protein contains several linear and conformational B cell epitopes, which suggests the antibody triggering property of this novel vaccine. Hence, our final protein can be used for prophylactic or therapeutic usages, because it can potentially stimulate both cellular and humoral immune responses. PMID:24512916

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

    PubMed

    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

  6. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

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

    2016-01-01

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

  7. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein.

    PubMed

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria. PMID:27194724

  8. Bäeklund Transformations, Conservation Laws and Linearization of the Self-Dual Yang-Mills and Chiral Fields

    SciTech Connect

    Wang, L. C.

    1980-01-01

    Bäecklund Transformations (BT) and the derivation of local conservation laws are first reviewed in the classic case of the Sine-Gordon equation. The BT, conservation laws (local and nonlocal), and the inverse-scattering formulation are discussed for the chiral and the self-dual Yang-Mills fields. Their possible applications to the loop formulation for the Yang-Mills fields are mentioned. 55 references, 1 figure.

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

    PubMed

    Oyarzun, P; Kobe, B

    2015-10-01

    Epitope-based vaccines (EVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant - immunogenic and conserved - antigen regions. Experimental screening of large sets of peptides is time-consuming and costly; therefore, in silico methods that facilitate T-cell epitope mapping of protein antigens are paramount for EV development. The prediction of T-cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T-cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T-cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state-of-the-art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens. PMID:26211755

  10. Functional, structural and epitopic prediction of hypothetical proteins of Mycobacterium tuberculosis H37Rv: An in silico approach for prioritizing the targets.

    PubMed

    Gazi, Md Amran; Kibria, Mohammad Golam; Mahfuz, Mustafa; Islam, Md Rezaul; Ghosh, Prakash; Afsar, Md Nure Alam; Khan, Md Arif; Ahmed, Tahmeed

    2016-10-15

    The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In

  11. Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin.

    PubMed

    Lainson, F A; Murray, J; Davies, R C; Donachie, W

    1996-09-01

    Defined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZ alpha fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes. PMID:8828217

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  14. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    PubMed Central

    Goh, Lucas Y. H.; Hobson-Peters, Jody; Prow, Natalie A.; Baker, Kelly; Piyasena, Thisun B. H.; Taylor, Carmel T.; Rana, Ashok; Hastie, Marcus L.; Gorman, Jeff J.; Hall, Roy A.

    2015-01-01

    Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP. PMID:26061335

  15. Immune recognition of citrullinated epitopes.

    PubMed

    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

  16. Efficient production of chimeric Human papillomavirus 16 L1 protein bearing the M2e influenza epitope in Nicotiana benthamiana plants

    PubMed Central

    2011-01-01

    Background Human papillomavirus 16 (HPV-16) L1 protein has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are highly immunogenic, allowing their use in vaccine production. Successful expression of HPV-16 L1 protein has been reported in plants, and plant-produced VLPs have been shown to be immunogenic after administration to animals. Results We investigated the potential of HPV-16 L1 to act as a carrier of two foreign epitopes from Influenza A virus: (i) M2e2-24, ectodomain of the M2 protein (M2e), that is highly conserved among all influenza A isolates, or (ii) M2e2-9, a shorter version of M2e containing the N-terminal highly conserved epitope, that is common for both M1 and M2 influenza proteins. A synthetic HPV-16 L1 gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the M2e2-24 or the M2e2-9 epitope in two predicted surface-exposed L1 positions. All chimeric constructs were transiently expressed in plants using the Cowpea mosaic virus-derived expression vector, pEAQ-HT. Chimeras were recognized by a panel of linear and conformation-specific anti HPV-16 L1 MAbs, and two of them also reacted with the anti-influenza MAb. Electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as VLPs of T = 1 or T = 7 symmetry, or capsomers. Conclusions In this study, we report for the first time the transient expression and the self-assembly of a chimeric HPV-16 L1 bearing the M2e influenza epitope in plants, representing also the first record of a successful expression of chimeric HPV-16 L1 carrying an epitope of a heterologous virus in plants. This study further confirms the usefulness of human papillomavirus particles as carriers of exogenous epitopes and their potential relevance for the production in plants of monovalent or multivalent vaccines. PMID:22085463

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

    PubMed

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

    2016-07-01

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

  18. Conservative finite volume solutions of a linear hyperbolic transport equation in two and three dimensions using multiple grids

    NASA Technical Reports Server (NTRS)

    Tiwari, Surendra N.; Kathong, Monchai

    1987-01-01

    The feasibility of the multiple grid technique is investigated by solving linear hyperbolic equations for simple two- and three-dimensional cases. The results are compared with exact solutions and those obtained from the single grid calculations. It is demonstrated that the technique works reasonably well when two grid systems contain grid cells of comparative sizes. The study indicates that use of the multiple grid does not introduce any significant error and that it can be used to attack more complex problems.

  19. The presence of prolines in the flanking region of an immunodominant HIV‐2 gag epitope influences the quality and quantity of the epitope generated

    PubMed Central

    Leligdowicz, Aleksandra; Kramer, Holger B.; Onyango, Clayton; Cotten, Matthew; Wright, Cynthia; Whittle, Hilton C.; McMichael, Andrew; Dong, Tao; Kessler, Benedikt M.; Rowland‐Jones, Sarah L.

    2015-01-01

    Both the recognition of HIV‐infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV‐2 gag‐specific T‐cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165DRFYKSLRA173, within the highly conserved Major Homology Region of gag‐p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag‐p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag‐specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165DRFYKSLRA173 epitope. Our data demonstrate that the 165DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral‐load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV‐2 encoding the “PPP‐gag” sequence and both strong gag‐specific CTL responses as well as lower viral load. PMID:26018465

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

    SciTech Connect

    Huang, Pi H.; Li, Ying J.; Su, Yu P.; Lee, Long H.; Liu, Hung J. . E-mail: hjliu@mail.npust.edu.tw

    2005-02-20

    We have previously shown that avian reovirus (ARV) {sigma}A and {sigma}NS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on {sigma}A (I and II) and three epitopes (A, B, and C) on {sigma}NS. To further define the location of epitopes on {sigma}A and {sigma}NS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of {sigma}A and {sigma}NS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on {sigma}A was located at amino acid residues {sup 340}QWVMAGLVSAA{sup 350} and epitope B on {sigma}NS at amino acid residues {sup 180}MLDMVDGRP{sup 188}. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of {sigma}A and {sigma}NS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete {sigma}NS but not with truncated {sigma}NS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on {sigma}NS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on {sigma}A was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the {sigma}NS with MAb 1F9. The {sigma}NS of ARV with ss

  1. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

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

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope. PMID:20362338

  2. Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

    PubMed Central

    Wu, Z; Kayman, S C; Honnen, W; Revesz, K; Chen, H; Vijh-Warrier, S; Tilley, S A; McKeating, J; Shotton, C; Pinter, A

    1995-01-01

    A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed

  3. An Unstable Th Epitope of P. falciparum Fosters Central Memory T Cells and Anti-CS Antibody Responses

    PubMed Central

    Parra-López, Carlos A.; Bernal-Estévez, David; Vargas, Luis Eduardo; Pulido-Calixto, Carolina; Salazar, Luz Mary; Calvo-Calle, J. Mauricio; Stern, Lawrence J.

    2014-01-01

    Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRβ1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed. PMID:24983460

  4. Epitope mapping by solution NMR spectroscopy.

    PubMed

    Bardelli, M; Livoti, E; Simonelli, L; Pedotti, M; Moraes, A; Valente, A P; Varani, L

    2015-06-01

    Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X-ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. PMID:25726811

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

    PubMed

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

    2016-01-14

    Mimics of discontinuous epitopes of for example bacterial or viral proteins may have considerable potential for the development of synthetic vaccines, especially if conserved epitopes can be mimicked. However, due to the structural complexity and size of discontinuous epitopes molecular construction of these mimics remains challeging. We present here a convergent route for the assembly of discontinuous epitope mimics by successive azide alkyne cycloaddition on an orthogonal alkyne functionalized scaffold. Here the synthesis of mimics of the HIV gp120 discontinuous epitope that interacts with the CD4 receptor is described. The resulting protein mimics are capable of inhibition of the gp120-CD4 interaction. The route is convergent, robust and should be applicable to other discontinuous epitopes. PMID:26552661

  6. Towards in silico prediction of immunogenic epitopes.

    PubMed

    Flower, Darren R

    2003-12-01

    As torrents of new data now emerge from microbial genomics, bioinformatic prediction of immunogenic epitopes remains challenging but vital. In silico methods often produce paradoxically inconsistent results: good prediction rates on certain test sets but not others. The inherent complexity of immune presentation and recognition processes complicates epitope prediction. Two encouraging developments - data driven artificial intelligence sequence-based methods for epitope prediction and molecular modeling methods based on three-dimensional protein structures - offer hope for the future. PMID:14644141

  7. Epitope topography controls bioactivity in supramolecular nanofibers

    PubMed Central

    Sur, Shantanu; Tantakitti, Faifan; Matson, John B.; Stupp, Samuel I.

    2015-01-01

    Incorporating bioactivity into artificial scaffolds using peptide epitopes present in the extracellular matrix (ECM) is a well-known approach. A common strategy has involved epitopes that provide cells with attachment points and external cues through interaction with integrin receptors. Although a variety of bioactive sequences have been identified so far, less is known about their optimal display in a scaffold. We report here on the use of self-assembled peptide amphiphile (PA) nanofiber matrices to investigate the impact of spatial presentation of the fibronectin derived epitope RGDS on cell response. Using one, three, or five glycine residues, RGDS epitopes were systematically spaced out from the surface of the rigid nanofibers. We found that cell morphology was strongly affected by the separation of the epitope from the nanofiber surface, with the longest distance yielding the most cell-spreading, bundling of actin filaments, and a round-to-polygonal transformation of cell shape. Cell response to this type of epitope display was also accompanied with activated integrin-mediated signaling and formation of stronger adhesions between cells and substrate. Interestingly, unlike length, changing the molecular flexibility of the linker had minimal influence on cell behavior on the substrate for reasons that remain poorly understood. The use in this study of high persistence length nanofibers rather than common flexible polymers allows us to conclude that epitope topography at the nanoscale structure of a scaffold influences its bioactive properties independent of epitope density and mechanical properties. PMID:25745558

  8. Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

    PubMed Central

    2010-01-01

    Background Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. Methods A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. Results Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. Conclusions This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations. PMID

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

    PubMed

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

    2015-04-01

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

  10. An analytical coupled technique for solving nonlinear large-amplitude oscillation of a conservative system with inertia and static non-linearity.

    PubMed

    Razzak, Md Abdur; Alam, Md Shamsul

    2016-01-01

    Based on a new trial function, an analytical coupled technique (a combination of homotopy perturbation method and variational method) is presented to obtain the approximate frequencies and the corresponding periodic solutions of the free vibration of a conservative oscillator having inertia and static non-linearities. In some of the previous articles, the first and second-order approximations have been determined by the same method of such nonlinear oscillator, but the trial functions have not been satisfied the initial conditions. It seemed to be a big shortcoming of those articles. The new trial function of this paper overcomes aforementioned limitation. The first-order approximation is mainly considered in this paper. The main advantage of this present paper is, the first-order approximation gives better result than other existing second-order harmonic balance methods. The present method is valid for large amplitudes of oscillation. The absolute relative error measures (first-order approximate frequency) in this paper is 0.00 % for large amplitude A = 1000, while the relative error gives two different second-order harmonic balance methods: 10.33 and 3.72 %. Thus the present method is suitable for solving the above-mentioned nonlinear oscillator. PMID:27119060

  11. Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

    PubMed

    Malito, Enrico; Faleri, Agnese; Lo Surdo, Paola; Veggi, Daniele; Maruggi, Giulietta; Grassi, Eva; Cartocci, Elena; Bertoldi, Isabella; Genovese, Alessia; Santini, Laura; Romagnoli, Giacomo; Borgogni, Erica; Brier, Sébastien; Lo Passo, Carla; Domina, Maria; Castellino, Flora; Felici, Franco; van der Veen, Stijn; Johnson, Steven; Lea, Susan M; Tang, Christoph M; Pizza, Mariagrazia; Savino, Silvana; Norais, Nathalie; Rappuoli, Rino; Bottomley, Matthew J; Masignani, Vega

    2013-02-26

    Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens. PMID:23396847

  12. The Immune Epitope Database 2.0

    PubMed Central

    Vita, Randi; Zarebski, Laura; Greenbaum, Jason A.; Emami, Hussein; Hoof, Ilka; Salimi, Nima; Damle, Rohini; Sette, Alessandro; Peters, Bjoern

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ∼99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009. PMID:19906713

  13. Unravelling viral camouflage: approaches to the study and characterization of conformational epitopes.

    PubMed

    Augustin, T; Cehlar, O; Skrabana, R; Majerova, P; Hanes, J

    2015-06-01

    Antibodies are broadly used in clinical and basic research. Many of monoclonal antibodies are successfully adopted for therapeutic and diagnostic targeting of viral pathogens. Efficacy of antiviral neutralizing or protective antibodies depends on their ability to recognize epitopes interfering with viral infection. However, viruses are able to incessantly change their antigenic determinants to escape surveillance of humoral immune system and therefore the successful antiviral therapies require continuous development. Characterization of interactions of antibodies with prevalently conformational viral epitopes is important for understanding antibody mode of action and can help to identify conserved regions that may be exploited in designing new vaccines and virus neutralizing antibodies. In this article, we are reviewing techniques in use for characterization of conformational epitopes of monoclonal antibodies with focus on viruses. PMID:26104327

  14. Prediction of B-Cell Epitopes in Listeriolysin O, a Cholesterol Dependent Cytolysin Secreted by Listeria monocytogenes

    PubMed Central

    Jones, Morris S.; Carter, J. Mark

    2014-01-01

    Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay. PMID:24523732

  15. Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

    PubMed

    Kundu, Prasun; Biswas, Rupam; Mukherjee, Somnath; Reinhard, Linda; Dutta, Anirudha; Mueller-Dieckmann, Jochen; Weiss, Manfred S; Pal, Nishit Kumar; Das, Amit Kumar

    2016-07-01

    Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel β strands stacked between α helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping. PMID:27189947

  16. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef.

    PubMed

    Milicic, Anita; Price, David A; Zimbwa, Peter; Booth, Bruce L; Brown, Helen L; Easterbrook, Philippa J; Olsen, Kara; Robinson, Nicola; Gileadi, Uzi; Sewell, Andrew K; Cerundolo, Vincenzo; Phillips, Rodney E

    2005-10-01

    CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated. PMID:16177107

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

    PubMed Central

    Depla, Erik; Van der Aa, Annegret; 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; Philip, Ramilla; Newman, Mark J.; Meheus, Lydie

    2008-01-01

    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 peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice. PMID:17942551

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

    PubMed Central

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

    2015-01-01

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

  19. Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

    PubMed Central

    Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O.; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen

    2013-01-01

    Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR. PMID:24244326

  20. Identification of B- and T-Cell Epitopes of BB, a Carrier Protein Derived from the G Protein of Streptococcus Strain G148

    PubMed Central

    Goetsch, Liliane; Haeuw, Jean Francois; Champion, Thierry; Lacheny, Christine; N’Guyen, Thien; Beck, Alain; Corvaia, Nathalie

    2003-01-01

    Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB. PMID:12522050

  1. Automatic Generation of Validated Specific Epitope Sets

    PubMed Central

    Carrasco Pro, Sebastian; Sidney, John; Paul, Sinu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro

    2015-01-01

    Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets. PMID:26568965

  2. Tools to MSn Sequence and Document the Structures of Glycan Epitopes

    PubMed Central

    Zhang, Hailong; Ashline, David J.

    2016-01-01

    Sequential disassembly (MSn) has been applied to fully characterise and document native samples containing glycan epitopes with their synthetic analogues. Both sample types were prepared by methylation, solvent phase extracted, directly infused and spatially resolved. Product ions of all samples were compiled and contrasted using management tools prepared for the fragment ion library. Each of the epitopes was further disassembled to confirm the multiple structural isomers probable within component substructures of linkage and branching. All native samples tested proved to be matched with their synthetic analogues and reasonably identical on either linear or cylindrical ion traps. Not surprisingly, spectra of mixed epitopes fragment independently, being uninfluenced by similarities. The approach has been coupled with computational tools for data handling and presentation. PMID:27110600

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

    PubMed Central

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

    1997-01-01

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

  4. Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes

    PubMed Central

    Crill, Wayne D.; Chang, Gwong-Jen J.

    2004-01-01

    The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231. PMID:15564505

  5. Cross-reactive human B cell and T cell epitopes between influenza A and B viruses

    PubMed Central

    2013-01-01

    Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4+ T cell epitopes conserved between the two genera were identified in humans. A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently. PMID:23886073

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

    PubMed

    Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa; Blixt, Ola; Arigi, Emma; Pereira, Daniela; Høgdall, Estrid; Mandel, Ulla; Bennett, Eric P; Vakhrushev, Sergey Y; David, Leonor; Clausen, Henrik

    2015-11-01

    The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes. PMID:26201951

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

    PubMed Central

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

    2013-01-01

    Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT. PMID:24222901

  8. Characterization of C-strain “Riems” TAV-epitope escape variants obtained through selective antibody pressure in cell culture

    PubMed Central

    2012-01-01

    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. PMID:22515281

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

    PubMed

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

    2015-11-27

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

  10. Mapping I-A(g7) restricted epitopes in murine G6PC2.

    PubMed

    Yang, Tao; Hohenstein, Anita C; Lee, Catherine E; Hutton, John C; Davidson, Howard W

    2013-03-01

    G6PC2, also known as islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP), is a major target of autoreactive CD8(+) T cells in both diabetic human subjects and the non-obese diabetic (NOD) mouse. However, in contrast to the abundant literature regarding the CD8(+) response to this antigen, much less is known about the potential involvement of IGRP-reactive CD4(+) T cells in diabetogenesis. The single previous study that examined this question in NOD mice was based upon a candidate epitope approach and identified three I-A(g7)-restricted epitopes that each elicited spontaneous responses in these animals. However, given the known inaccuracies of MHC class II epitope prediction algorithms, we hypothesized that additional specificities might also be targeted. To address this issue, we immunized NOD mice with membranes from insect cells overexpressing full-length recombinant mouse IGRP and measured recall responses of purified CD4(+) T cells using a library of overlapping peptides encompassing the entire 355-aa primary sequence. Nine peptides representing 8 epitopes gave recall responses, only 1 of which corresponded to any of the previously reported sequences. In each case proliferation was blocked by a monoclonal antibody to I-A(g7), but not the appropriate isotype control. Consistent with a role in diabetogenesis, proliferative responses to 4 of the 9 peptides (3 epitopes) were also detected in CD4(+) T cells purified from the pancreatic draining lymph nodes of pre-diabetic female animals, but not from peripheral lymph nodes or spleens of the same animals. Intriguingly, one of the newly identified spontaneously reactive epitopes (P8 [IGRP(55-72)]) is highly conserved between mice and man, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects, an hypothesis that we are currently testing. PMID:22983906

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

    PubMed Central

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

    2014-01-01

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

  12. Design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach

    PubMed Central

    Oany, Arafat Rahman; Emran, Abdullah-Al; Jyoti, Tahmina Pervin

    2014-01-01

    Human coronavirus (HCoV), a member of Coronaviridae family, is the causative agent of upper respiratory tract infections and “atypical pneumonia”. Despite severe epidemic outbreaks on several occasions and lack of antiviral drug, not much progress has been made with regard to an epitope-based vaccine designed for HCoV. In this study, a computational approach was adopted to identify a multiepitope vaccine candidate against this virus that could be suitable to trigger a significant immune response. Sequences of the spike proteins were collected from a protein database and analyzed with an in silico tool, to identify the most immunogenic protein. Both T cell immunity and B cell immunity were checked for the peptides to ensure that they had the capacity to induce both humoral and cell-mediated immunity. The peptide sequence from 88–94 amino acids and the sequence KSSTGFVYF were found as the most potential B cell and T cell epitopes, respectively. Furthermore, conservancy analysis was also done using in silico tools and showed a conservancy of 64.29% for all epitopes. The peptide sequence could interact with as many as 16 human leukocyte antigens (HLAs) and showed high cumulative population coverage, ranging from 75.68% to 90.73%. The epitope was further tested for binding against the HLA molecules, using in silico docking techniques, to verify the binding cleft epitope interaction. The allergenicity of the epitopes was also evaluated. This computational study of design of an epitope-based peptide vaccine against HCoVs allows us to determine novel peptide antigen targets in spike proteins on intuitive grounds, albeit the preliminary results thereof require validation by in vitro and in vivo experiments. PMID:25187696

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

    PubMed

    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

  14. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

    PubMed

    Ebstein, F; Textoris-Taube, K; Keller, C; Golnik, R; Vigneron, N; Van den Eynde, B J; Schuler-Thurner, B; Schadendorf, D; Lorenz, F K M; Uckert, W; Urban, S; Lehmann, A; Albrecht-Koepke, N; Janek, K; Henklein, P; Niewienda, A; Kloetzel, P M; Mishto, M

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  15. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  16. Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP)

    PubMed Central

    2013-01-01

    Background Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen. Methods Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence. Results Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A

  17. Differential Recognition of Influenza A Viruses by M158–66 Epitope-Specific CD8+ T Cells Is Determined by Extraepitopic Amino Acid Residues

    PubMed Central

    van de Sandt, Carolien E.; Kreijtz, Joost H. C. M.; Geelhoed-Mieras, Martina M.; Nieuwkoop, Nella J.; Spronken, Monique I.; van de Vijver, David A. M. C.; Fouchier, Ron A. M.; Osterhaus, Albert D. M. E.

    2015-01-01

    ABSTRACT Natural influenza A virus infections elicit both virus-specific antibody and CD4+ and CD8+ T cell responses. Influenza A virus-specific CD8+ cytotoxic T lymphocytes (CTLs) contribute to clearance of influenza virus infections. Viral CTL epitopes can display variation, allowing influenza A viruses to evade recognition by epitope-specific CTLs. Due to functional constraints, some epitopes, like the immunodominant HLA-A*0201-restricted matrix protein 1 (M158–66) epitope, are highly conserved between influenza A viruses regardless of their subtype or host species of origin. We hypothesized that human influenza A viruses evade recognition of this epitope by impairing antigen processing and presentation by extraepitopic amino acid substitutions. Activation of specific T cells was used as an indication of antigen presentation. Here, we show that the M158–66 epitope in the M1 protein derived from human influenza A virus was poorly recognized compared to the M1 protein derived from avian influenza A virus. Furthermore, we demonstrate that naturally occurring variations at extraepitopic amino acid residues affect CD8+ T cell recognition of the M158–66 epitope. These data indicate that human influenza A viruses can impair recognition by M158–66-specific CTLs while retaining the conserved amino acid sequence of the epitope, which may represent a yet-unknown immune evasion strategy for influenza A viruses. This difference in recognition may have implications for the viral replication kinetics in HLA-A*0201 individuals and spread of influenza A viruses in the human population. The findings may aid the rational design of universal influenza vaccines that aim at the induction of cross-reactive virus-specific CTL responses. IMPORTANCE Influenza viruses are an important cause of acute respiratory tract infections. Natural influenza A virus infections elicit both humoral and cellular immunity. CD8+ cytotoxic T lymphocytes (CTLs) are directed predominantly against

  18. Newcastle Disease Virus (NDV) Marker Vaccine: an Immunodominant Epitope on the Nucleoprotein Gene of NDV Can Be Deleted or Replaced by a Foreign Epitope

    PubMed Central

    Mebatsion, Teshome; Koolen, Marck J. M.; de Vaan, Leonie T. C.; de Haas, Niels; Braber, Marian; Römer-Oberdörfer, Angela; van den Elzen, Paul; van der Marel, Pieter

    2002-01-01

    The nucleoprotein (NP) of Newcastle disease virus (NDV) functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens. Here, we localized a conserved B-cell immunodominant epitope (IDE) spanning residues 447 to 455 and successfully generated a recombinant NDV lacking the IDE by reverse genetics. Despite deletion of NP residues 443 to 460 encompassing the NP-IDE, the mutant NDV propagated in embryonated specific-pathogen-free chicken eggs to a level comparable to that of the parent virus. In addition, a B-cell epitope of the S2 glycoprotein of murine hepatitis virus (MHV) was inserted in-frame to replace the NP-IDE. Recombinant viruses properly expressing the introduced MHV epitope were successfully generated, demonstrating that the NP-IDE not only is dispensable for virus replication but also can be replaced by foreign sequences. Chickens immunized with the hybrid recombinants produced specific antibodies against the S2 glycoprotein of MHV and completely lacked antibodies directed against the NP-IDE. These marked-NDV recombinants, in conjunction with a diagnostic test, enable serological differentiation of vaccinated animals from infected animals and may be useful tools in ND eradication programs. The identification of a mutation-permissive region on the NP gene allows a rational approach to the insertion of protective epitopes and may be relevant for the design of NDV-based cross-protective marker vaccines. PMID:12239288

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

    Umair, Saleh; Deng, Qing; Roberts, Joanna M; Shaw, Richard J; Sutherland, Ian A; Pernthaner, Anton

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  5. Production, Characterization, and Epitope Mapping of Monoclonal Antibodies Against Different Subtypes of Rabbit Hemorrhagic Disease Virus (RHDV)

    PubMed Central

    Kong, Desheng; Liu, Jiasen; Jiang, Qian; Yu, Zuo; Hu, Xiaoliang; Guo, Dongchun; Huang, Qianqian; Jiao, Meihui; Qu, Liandong

    2016-01-01

    In 2010, a new rabbit hemorrhagic disease virus (RHDV) variant, designated RHDV2, was identified for the first time in Italy. Studies have shown that RHDV2 differs from RHDV1 (traditional RHDV) in terms of its antigenic profile and genetic characteristics. The VP60 protein of RHDV is a structural protein that plays important roles in viral replication, assembly, and immunogenicity. In this study, we immunized BALB/c mice with recombinant VP60 proteins from different RHDV subtypes. After three rounds of subcloning, type-specific positive hybridoma clones of RHDV1 and RHDV2 were further identified by an enzyme-linked immunosorbent assay, Western blotting, and an indirect immunofluorescence assay. Finally, three monoclonal antibodies (MAbs) (1D6, 1H2, and 3F2) that only recognize RHDV1, and four MAbs (1G2, 2C1, 3B7, and 5D6) that only recognize RHDV2 were identified. The epitopes recognized by these MAbs were mapped by Western blotting. Sequence analysis showed that the epitope sequences recognized by 1D6, 1H2, and 3F2 are highly conserved (98%) among RHDV1 strains, whereas the epitope sequences recognized by 1G2, 2C1, 3B7, and 5D6 are 100% conserved among RHDV2 strains. The high conservation of the epitope sequence showed that the screened MAbs were type-specific, and that they could distinguish different RHDV subtypes. PMID:26878800

  6. Design and Antigenic Epitopes Prediction of a New Trial Recombinant Multiepitopic Rotaviral Vaccine: In Silico Analyses.

    PubMed

    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

  7. Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories

    PubMed Central

    Otsu, K.; Guo, R.; Dreskin, S. C.

    2015-01-01

    Summary Background Ara h 2 and Ara h 6 are moderately homologous and highly potent peanut allergens. Objective To identify IgE-binding linear epitopes of Ara h 6, compare them to those of Ara h 2, and to stratify binding based on clinical histories. Methods Thirty highly peanut-allergic subjects were stratified by clinical history. Sera were diluted to contain the same amount of anti-peanut IgE. IgE binding to overlapping 20-mer peptides of Ara h 2 and Ara h 6 was assessed using microarrays. Results Each subject had a unique IgE-binding fingerprint to peptides; these data were coalesced into epitope binding. IgE from subjects with a history of more severe reactions (n = 19) had a smaller frequency of binding events (BEs) for both Ara h 2 (52 BEs of 152 (19×8epitopes) possible BEs and Ara h 6 (13 BEs of 133 (19×7 epitopes) possible BEs) compared to IgE from those with milder histories (n = 11) (Ara h 2: 47 BEs of 88 (11×8 epitopes) possible BEs, P < 0.01; Ara h 6: 25 BEs of 77 (11×7 epitopes) possible BEs, P < 0.001). Using an unsupervised hierarchal cluster analysis, subjects with similar histories tended to cluster. We have tentatively identified a high-risk pattern of binding to peptides of Ara h 2 and Ara h 6, predominantly in subjects with a history of more severe reactions (OR = 12.6; 95% CI: 2.0–79.5; P < 0.01). Conclusions and Clinical Relevance IgE from patients with more severe clinical histories recognize fewer linear epitopes of Ara h 2 and Ara h 6 than do subjects with milder reactions and bind these epitopes in characteristic patterns. Close examination of IgE binding to epitopes of Ara h 2 and Ara h 6 may have prognostic value. PMID:25213872

  8. Expression and immune response to foreign epitopes in bacteria. Perspectives for live vaccine development.

    PubMed

    Charbit, A; Newton, S M; Klebba, P E; Clément, J M; Fayolle, C; Lo-Man, R; Leclerc, C; Hofnung, M

    1997-02-01

    We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different regions of a carrier protein. By using as carriers, two bacterial envelope proteins, the LamB and MalE proteins of E. coli K12, we were able to express the same epitope in different sites of the two proteins and in different compartments of the bacteria. This allowed us to analyze the influence of the localization in E. coli cells of a foreign B-cell epitope on the induction of specific antibody responses, and the role of the molecular environment on the immunological properties of foreign B- or T-cell epitopes, using either purified hybrid proteins or live recombinant bacteria. Several LamB and MalE hybrid proteins were expressed in the aroA attenuated strain of S. typhimurium, SL3261. Immunizations of mice with live recombinant bacteria by the intravenous route showed that it was possible to induce humoral responses against inserted foreign sequences. In order to improve the in vivo stability of the plasmids carrying the different contructions, and to increase the amounts of recombinant LamB and MalE hybrid proteins expressed in vivo, the LamB and malE genes were placed under the control of the anaerobically inducible pnirBpromoter control. The genetic factors susceptible of influencing the immune response to recombinant Salmonella in mice were also studied. PMID:9382734

  9. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  10. Epitope specificity of human immunodeficiency virus-1 antibody dependent cellular cytotoxicity [ADCC] responses.

    PubMed

    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

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

    PubMed Central

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

    2010-01-01

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

  12. Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity

    PubMed Central

    Keskin, Derin B.; Reinhold, Bruce B.; Zhang, Guang Lan; Ivanov, Alexander R.; Karger, Barry L.; Reinherz, Ellis L.

    2015-01-01

    Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC50 < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M13–11 to a high of >500 for M158–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M158–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M158–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M158–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery. PMID:25646416

  13. Identification and retrospective validation of T-cell epitopes in the hepatitis C virus genotype 4 proteome

    PubMed Central

    Abdel-Hady, Karim M; Gutierrez, Andres H; Terry, Frances; Desrosiers, Joe; De Groot, Anne S; Azzazy, Hassan ME

    2014-01-01

    With over 150 million people chronically infected worldwide and millions more infected annually, hepatitis C continues to pose a burden on the global healthcare system. The standard therapy of hepatitis C remains expensive, with severe associated side effects and inconsistent cure rates. Vaccine development against the hepatitis C virus has been hampered by practical and biological challenges posed by viral evasion mechanisms. Despite these challenges, HCV vaccine research has presented a number of candidate vaccines that progressed to phase II trials. However, those efforts focused mainly on HCV genotypes 1 and 2 as vaccine targets and barely enough attention was given to genotype 4, the variant most prevalent in the Middle East and central Africa. We describe herein the in silico identification of highly conserved and immunogenic T-cell epitopes from the HCV genotype 4 proteome, using the iVAX immunoinformatics toolkit, as targets for an epitope-driven vaccine. We also describe a fast and inexpensive approach for results validation using the empirical data on the Immune Epitope Database (IEDB) as a reference. Our analysis identified 90 HLA class I epitopes of which 20 were found to be novel and 19 more had their binding predictions retrospectively validated; empirical data for the remaining 51 epitopes was insufficient to validate their binding predictions. Our analysis also identified 14 HLA class II epitopes, of which 8 had most of their binding predictions validated. Further investigation is required regarding the efficacy of the identified epitopes as vaccine targets in populations where HCV genotype 4 is most prevalent. PMID:25424944

  14. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    SciTech Connect

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  15. Distribution of lipophosphoglycan-associated epitopes in different Leishmania species and in African trypanosomes.

    PubMed

    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

  16. Characterization of specific antigenic epitopes and the nuclear export signal of the Porcine circovirus 2 ORF3 protein.

    PubMed

    Gu, Jinyan; Wang, Lun; Jin, Yulan; Lin, Cui; Wang, Huijuan; Zhou, Niu; Xing, Gang; Liao, Min; Zhou, Jiyong

    2016-02-29

    Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, we find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3. PMID:26854343

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

    PubMed Central

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

    2016-01-01

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

  18. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    PubMed

    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

  19. In silico prediction and ex vivo evaluation of potential T-cell epitopes in glycoproteins 4 and 5 and nucleocapsid protein of genotype-I (European) of porcine reproductive and respiratory syndrome virus.

    PubMed

    Díaz, Ivan; Pujols, Joan; Ganges, Llilianne; Gimeno, Mariona; Darwich, Laila; Domingo, Mariano; Mateu, Enric

    2009-09-18

    T-cell epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) glycoproteins 4 (GP4), 5 (GP5) and nucleocapsid (N) were predicted using bioinformatics and later tested by IFN-gamma ELISPOT in pigs immunized with either a modified live vaccine (MLV) or DNA (open reading frames 4, 5 or 7). For MLV-vaccinated pigs, immunodominant epitopes were found in N but T-epitopes were also found in GP4 and GP5. For DNA-immunized pigs, some peptides were differently recognized. Using a large set of PRRSV sequences it was shown that N contains a conserved epitope and that for GP5, the genotype-I counterparts of previously reported epitopes of genotype-II strains were also immunogenic. PMID:19646408

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

    PubMed

    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

  1. Advances in the study of HLA-restricted epitope vaccines

    PubMed Central

    Zhao, Lingxiao; Zhang, Min; Cong, Hua

    2013-01-01

    Vaccination is a proven strategy for protection from disease. An ideal vaccine would include antigens that elicit a safe and effective protective immune response. HLA-restricted epitope vaccines, which include T-lymphocyte epitopes restricted by HLA alleles, represent a new and promising immunization approach. In recent years, research in HLA-restricted epitope vaccines for the treatment of tumors and for the prevention of viral, bacterial, and parasite-induced infectious diseases have achieved substantial progress. Approaches for the improvement of the immunogenicity of epitope vaccines include (1) improving the accuracy of the methods used for the prediction of epitopes, (2) making use of additional HLA-restricted CD8+ T-cell epitopes, (3) the inclusion of specific CD4+ T-cell epitopes, (4) adding B-cell epitopes to the vaccine construction, (5) finding more effective adjuvants and delivery systems, (6) using immunogenic carrier proteins, and (7) using multiple proteins as epitopes sources. In this manuscript, we review recent research into HLA-restricted epitope vaccines. PMID:23955319

  2. Characterization of continuous B-cell epitopes in the N-terminus of glutamate decarboxylase67 using monoclonal antibodies.

    PubMed

    Agca, Selin; Houen, Gunnar; Trier, Nicole Hartwig

    2014-12-01

    Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type-1 diabetes (T1D) and stiff-person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ-aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme-linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N-terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14-23 and 91-99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side-chain functionality, rather than amino acid side-chain specificity. Additionally, results suggested that non-contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide-based therapeutics against SPS. PMID:25358241

  3. Probing the Impact of Local Structural Dynamics of Conformational Epitopes on Antibody Recognition.

    PubMed

    Liang, Yu; Guttman, Miklos; Davenport, Thaddeus M; Hu, Shiu-Lok; Lee, Kelly K

    2016-04-19

    Antibody-antigen interactions are governed by recognition of specific residues and structural complementarity between the antigen epitope and antibody paratope. While X-ray crystallography has provided detailed insights into static conformations of antibody-antigen complexes, factors such as conformational flexibility and dynamics, which are not readily apparent in the structures, can also have an impact on the binding event. Here we investigate the contribution of dynamics in the HIV-1 gp120 glycoprotein to antibody recognition of conserved conformational epitopes, including the CD4- and coreceptor-binding sites, and an inner domain site that is targeted by ADCC-active antibodies. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) was used to measure local structural dynamics across a panel of variable loop truncation mutants of HIV-1 gp120, including full-length gp120, ΔV3, ΔV1/V2, and extended core, which includes ΔV1/V2 and V3 loop truncations. CD4-bound full-length gp120 was also examined as a reference state. HDX-MS revealed a clear trend toward an increased level of order of the conserved subunit core resulting from loop truncation. Combined with biolayer interferometry and enzyme-linked immunosorbent assay measurements of antibody-antigen binding, we demonstrate that an increased level of ordering of the subunit core was associated with better recognition by an array of antibodies targeting complex conformational epitopes. These results provide detailed insight into the influence of structural dynamics on antibody-antigen interactions and suggest the importance of characterizing the structural stability of vaccine candidates to improve antibody recognition of complex epitopes. PMID:27003615

  4. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

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

    PubMed Central

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

    2014-01-01

    Middle East Respiratory Syndrome Coronavirus (MERS-CoV) belongs to the coronaviridae family. In spite of several outbreaks in the very recent years, no vaccine against this deadly virus is developed yet. In this study, the receptor binding domain (RBD) of Spike (S) glycoprotein of MERS-CoV was analyzed through Computational Immunology approach to identify the antigenic determinants (epitopes). In order to do so, the sequences of S glycoprotein that belong to different geographical regions were aligned to observe the conservancy of MERS-CoV RBD. The immune parameters of this region were determined using different in silico tools and Immune Epitope Database (IEDB). Molecular docking study was also employed to check the affinity of the potential epitope towards the binding cleft of the specific HLA allele. The N-terminus RBD (S367-S606) of S glycoprotein was found to be conserved among all the available strains of MERS-CoV. Based on the lower IC50 value, a total of eight potential T-cell epitopes and 19 major histocompatibility complex (MHC) class-I alleles were identified for this conserved region. A 9-mer epitope CYSSLILDY displayed interactions with the maximum number of MHC class-I molecules and projected the highest peak in the B-cell antigenicity plot which concludes that it could be a better choice for designing an epitope based peptide vaccine against MERSCoV considering that it must undergo further in vitro and in vivo experiments. Moreover, in molecular docking study, this epitope was found to have a significant binding affinity of -8.5 kcal/mol towards the binding cleft of the HLA-C*12:03 molecule. PMID:25258490

  6. In vivo protection against Tityus serrulatus scorpion venom by antibodies raised against a discontinuous synthetic epitope.

    PubMed

    Duarte, Clara Guerra; Alvarenga, Larissa Magalhães; Dias-Lopes, Camila; Machado-de-Avila, Ricardo Andrés; Nguyen, Christophe; Molina, Frank; Granier, Claude; Chávez-Olórtegui, Carlos

    2010-02-01

    Scorpion stings cause human fatalities in numerous countries. Serotherapy is the only specific means to try to circumvent the noxious effects of venom toxins. TsNTxP is a natural anatoxin from the venom of the scorpion Tityus serrulatus that may be useful to raise therapeutic anti-venom sera. Linear epitopes recognized by anti-TsNTxP antibodies have previously been mapped. Here, we attempted to identify discontinuous epitopes in TsNTxP since neutralizing epitopes are often associated with such complex entities. One hundred and fifty-three octadecapeptides with the general formula (P1)-(Gly-Gly)-(P2) were synthesized by the Spot method on cellulose membranes. P1 and P2 were octapeptides from the TsNTxP N-terminal and C-terminal sections, respectively. Each sequence of eight amino acids was frameshifted in turn by three residues, in order to cover TsNTxP entire sequence. Binding of neutralizing anti-TsNTxP rabbit antibodies to spotted peptides revealed GREGYPADGGGLPDSVKI as the more reactive peptide sequence. This epitope was made from the first eight residues of the protein (GREGYPAD) and from residues 47 to 54 (GLPDSVKI) of the C-terminal part of TsNTxP. BALB/c mice were immunized with synthetic GREGYPADGGGLPDSVKI peptide conjugated to ovalbumin. One week after the last immunization, in vivo protection assays showed that immunized mice could resist a challenge by an amount of T.serrulatus whole venom equivalent to 1.75 LD(100), a dose that killed all control non-immune mice. Based on molecular models of TsNTxP and related Tityus toxins, we found that the above peptide matches with a discontinuous epitope, well exposed at the toxin molecular surface which contains residues known to be important for the bioactivity of toxins. PMID:19948263

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

    PubMed Central

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

    1994-01-01

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

  8. Flexible vs Rigid Epitope Conformations for Diagnostic- and Vaccine-Oriented Applications: Novel Insights from the Burkholderia pseudomallei BPSL2765 Pal3 Epitope.

    PubMed

    Gori, Alessandro; Peri, Claudio; Quilici, Giacomo; Nithichanon, Arnone; Gaudesi, Davide; Longhi, Renato; Gourlay, Louise; Bolognesi, Martino; Lertmemongkolchai, Ganjana; Musco, Giovanna; Colombo, Giorgio

    2016-03-11

    Peptides seldom retain stable conformations if separated from their native protein structure. In an immunological context, this potentially affects the development of selective peptide-based bioprobes and, from a vaccine perspective, poses inherent limits in the elicitation of cross-reactive antibodies by candidate epitopes. Here, a 1,4-disubstituted-1,2,3-triazole-mediated stapling strategy was used to stabilize the native α-helical fold of the Pal3 peptidic epitope from the protein antigen PalBp (BPSL2765) from Burkholderia pseudomallei, the etiological agent of melioidosis. Whereas Pal3 shows no propensity to fold outside its native protein context, the engineered peptide (Pal3H) forms a stable α-helix, as assessed by MD, NMR, and CD structural analyses. Importantly, Pal3H shows an enhanced ability to discriminate between melioidosis patient subclasses in immune sera reactivity tests, demonstrating the potential of the stapled peptide for diagnostic purposes. With regard to antibody elicitation and related bactericidal activities, the linear peptide is shown to elicit a higher response. On these bases, we critically discuss the implications of epitope structure engineering for diagnostic- and vaccine-oriented applications. PMID:27623032

  9. Fine mapping of canine parvovirus B cell epitopes.

    PubMed

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

    1991-10-01

    In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization. PMID:1919526

  10. Introducing Conservation of Momentum

    ERIC Educational Resources Information Center

    Brunt, Marjorie; Brunt, Geoff

    2013-01-01

    The teaching of the principle of conservation of linear momentum is considered (ages 15 + ). From the principle, the momenta of two masses in an isolated system are considered. Sketch graphs of the momenta make Newton's laws appear obvious. Examples using different collision conditions are considered. Conservation of momentum is considered…

  11. Proof of principle for epitope-focused vaccine design

    PubMed Central

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

    2014-01-01

    Summary Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Multiple major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus (RSV), that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for research and development of a human RSV vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets including antigenically highly variable pathogens such as HIV and influenza. PMID:24499818

  12. Proof of principle for epitope-focused vaccine design

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  13. Transcriptome analysis of Solanum melongena L. (eggplant) fruit to identify putative allergens and their epitopes.

    PubMed

    Ramesh, Kumar Ramagoni; Hemalatha, R; Vijayendra, Chary Anchoju; Arshi, Uz Zaman Syed; Dushyant, Singh Baghel; Dinesh, Kumar Bharadwaj

    2016-01-15

    Eggplant is the third most important Solanaceae crop after tomato and potato, particularly in India and China. A transcriptome analysis of eggplant's fruit was performed to study genes involved in medicinal importance and allergies. Illumina HiSeq 2000 system generated 89,763,638 raw reads (~18 Gb) from eggplant. High quality reads (59,039,694) obtained after trimming process, were assembled into a total of 149,224 non redundant set of transcripts. Out of 80,482 annotated sequences of eggplant fruit (BLASTx results against nr-green plant database), 40,752 transcripts showed significant similarity with predicted proteins of Solanum tuberosum (51%) followed by Solanum lycopersicum (34%) and other sequenced plant genomes. With BLASTx top hit analysis against existing allergens, a total of 1986 homologous allergen sequences were found, which had >37% similarity with 48 different allergens existing in the database. From the 48 putative allergens, 526 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. Transcript sequences generated from this study can be used to map epitopes of monoclonal antibodies and polyclonal sera from patients. With the support of this whole transcriptome catalogue of eggplant fruit, complete list of genes can be predicted based on which secondary structures of proteins may be modeled. PMID:26424595

  14. De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes

    PubMed Central

    Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

    2015-01-01

    Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37–100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

  15. B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

    PubMed Central

    Pancino, G; Chappey, C; Saurin, W; Sonigo, P

    1993-01-01

    In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines. Images PMID:7678301

  16. De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes.

    PubMed

    Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

    2015-01-01

    Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

  17. Atomic-level mapping of antibody epitopes on a GPCR.

    PubMed

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry. PMID:19453194

  18. Recognition of dengue virus protein using epitope-mediated molecularly imprinted film.

    PubMed

    Tai, Dar-Fu; Lin, Chung-Yin; Wu, Tzong-Zeng; Chen, Li-Kuang

    2005-08-15

    Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required. PMID:16097751

  19. Sequence-based in silico analysis of well studied Hepatitis C Virus epitopes and their variants in other genotypes (particularly genotype 5a) against South African human leukocyte antigen backgrounds

    PubMed Central

    2012-01-01

    Background Host genetics influence the outcome of HCV disease. HCV is also highly mutable and escapes host immunity. HCV genotypes are geographically distributed and HCV subtypes have been shown to have distinct repertoires of HLA-restricted viral epitopes which explains the lack of cross protection across genotypes observed in some studies. Despite this, immune databases and putative epitope vaccines concentrate almost exclusively on HCV genotype 1 class I-epitopes restricted by the HLA-A*02 allele. While both genotype and allele predominate in developed countries, we hypothesise that HCV variation and population genetics will affect the efficacy of proposed epitope vaccines in South Africa. This in silico study investigates HCV viral variability within well-studied epitopes identified in genotype 1 and uses algorithms to predict the immunogenicity of their variants from other less studied genotypes and thus rate the most promising vaccine candidates for the South African population. Six class I- and seven class II- restricted epitope sequences within the core, NS3, NS4B and NS5B regions were compared across the six HCV genotypes using local genotype 5a sequence data together with global data. Common HLA alleles in the South African population are A30:01, A02:01, B58:02, B07:02; DRB1*13:01 and DRB1*03:01. Epitope binding to 13 class I- and 8 class –II alleles were described using web-based prediction servers, Immune Epitope Database, (IEDB) and Propred. Online population coverage tools were used to assess vaccine efficacy. Results Despite the homogeneity of genotype 1 and genotype 5 over the epitopes, there was limited promiscuity to local HLA-alleles.Host differences will make a putative vaccine less effective in South Africa. Of the 6 well-characterized class I- epitopes, only 2 class I- epitopes were promiscuous and 3 of the 7 class-II epitopes were better conserved and promiscuous. By fine tuning the putative vaccine using an optimal cocktail of genotype 1

  20. Identification of CD8 T cell epitopes in VP2 and NS1 proteins of African horse sickness virus in IFNAR(-/-) mice.

    PubMed

    de la Poza, Francisco; Marín-López, Alejandro; Castillo-Olivares, Javier; Calvo-Pinilla, Eva; Ortego, Javier

    2015-12-01

    African horse sickness virus (AHSV) is an Orbivirus of the family Reoviridae that causes severe pathology in equids. Previous work in our laboratory showed the presence of AHSV-specific CD8(+) T cells in mice immunized with recombinant Modified Vaccinia Ankara (rMVA) expressing VP2 and NS1 proteins. In the present work, we selected potential CD8 T cell epitopes (MHC-class I binding peptides) for the 129 mouse strain from the VP2 and NS1 proteins of AHSV-4, using a combination of four epitope prediction algorithms (SYFPEITHI, BYMAS, NetMHC I and NetMHCpan). ELISPOT and Intracellular Cytokine Staining (ICS) analysis showed that the VP2-720 (MSLLNFGAV), VP2-1044 (YTFGNKFLL), and NS1-83 (CVIKNADYV) peptides elicited IFN-γ production in splenocytes of MVA-VP2 and MVA-NS1 immunized mice and were identified as CD8(+) T cell epitopes. In addition, these three MHC-class I-binding peptides induced the expression of CD107a in CD8(+) T cells, an indirect marker of cytotoxic activity. Importantly, VP2-1044 and NS1-83 epitopes are conserved among all nine AHSV serotypes. These data demonstrate the activation of AHSV specific T-cell epitopes during vaccination with rMVAs expressing VP2 and NS1. Furthermore, the characterization of these CD8(+) T-cell epitopes provides information useful for the design of novel marker multiserotype vaccines against AHSV. PMID:26272673

  1. Evaluation of the use of non-pathogenic porcine circovirus type 1 as a vaccine delivery virus vector to express antigenic epitopes of porcine reproductive and respiratory syndrome virus.

    PubMed

    Piñeyro, Pablo E; Kenney, Scott P; Giménez-Lirola, Luis G; Opriessnig, Tanja; Tian, Debin; Heffron, C Lynn; Meng, Xiang-Jin

    2016-02-01

    We previously demonstrated that the C-terminus of the capsid gene of porcine circovirus type 2 (PCV2) is an immune reactive epitope displayed on the surface of virions. Insertion of foreign epitope tags in the C-terminus produced infectious virions that elicited humoral immune responses against both PCV2 capsid and the inserted epitope tags, whereas mutation in the N terminus impaired viral replication. Since the non-pathogenic porcine circovirus type 1 (PCV1) shares similar genomic organization and significant sequence identity with pathogenic PCV2, in this study we evaluated whether PCV1 can serve as a vaccine delivery virus vector. Four different antigenic determinants of porcine reproductive and respiratory syndrome virus (PRRSV) were inserted in the C-terminus of the PCV1 capsid gene, the infectivity and immunogenicity of the resulting viruses are determined. We showed that an insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not affect PCV1 replication. We successfully rescued and characterized four chimeric PCV1 viruses expressing PRRSV linear antigenic determinants (GP2 epitope II: aa 40-51, ASPSHVGWWSFA; GP3 epitope I: aa 61-72, QAAAEAYEPGRS; GP5 epitope I: aa 35-46, SSSNLQLIYNLT; and GP5 epitope IV: aa 187-200, TPVTRVSAEQWGRP). We demonstrated that all chimeric viruses were stable and infectious in vitro and three chimeric viruses were infectious in vivo. An immunogenicity study in pigs revealed that PCV1-VR2385EPI chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. The results have important implications for further evaluating PCV1 as a potential vaccine delivery vector. PMID:26555162

  2. Gag Protein Epitopes Recognized by CD4+ T-Helper Lymphocytes from Equine Infectious Anemia Virus-Infected Carrier Horses

    PubMed Central

    Lonning, S. M.; Zhang, W.; McGuire, T. C.

    1999-01-01

    Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV. PMID:10196322

  3. The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin

    PubMed Central

    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

  4. Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin

    PubMed Central

    Bakeberg, Jason L.; Tammachote, Rachaneekorn; Woollard, John R.; Hogan, Marie C.; Tuan, Han-Fang; Li, Ming; van Deursen, Jan M.; Wu, Yanhong; Huang, Bing Q.; Torres, Vicente E.; Harris, Peter C.

    2011-01-01

    Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15–550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin. PMID:22021705

  5. The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin.

    PubMed

    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

  6. Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin.

    PubMed

    Hu, Jin-Fang; Sun, Chun-Yun; Rao, Mu-Ding; Xie, Liang-Zhi

    2016-08-01

    Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection. PMID:27594152

  7. COMPETITIVE ENZYME-LINKED IMMUNOSORBENT ASSAY BASED ON A RHOPTRY-ASSOCIATED PROTEIN 1 EPITOPE SPECIFICALLY IDENTIFIES BABESIA BOVIS-INFECTED CATTLE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The competitive ELISA (cELISA) format has proven to be an accurate, reliable, easily standardized, and high throughput method to detect hemoparasite infections. In this study, a specied-specific, broadly conserved, and tandemly repeated B-cell epitope within the C-terminus of the rhoptry associated ...

  8. 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

    PubMed Central

    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

  9. The identification and characterization of epitopes in the 30-34 kDa Trypanosoma cruzi proteins recognized by antibodies in the serum samples of chagasic patients.

    PubMed

    Verissimo da Costa, Giovani Carlo; Lery, Leticia Miranda Santos; da Silva, Manuela Leal; Moura, Hércules; Peralta, Regina Helena Saramago; von Krüger, Wanda Maria Almeida; Bisch, Paulo Mascarello; Barr, John R; Peralta, José Mauro

    2013-03-27

    Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools. PMID:23159400

  10. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

  11. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

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

  12. Bioinformatics analysis of the epitope regions for norovirus capsid protein

    PubMed Central

    2013-01-01

    Background Norovirus is the major cause of nonbacterial epidemic gastroenteritis, being highly prevalent in both developing and developed countries. Despite of the available monoclonal antibodies (MAbs) for different sub-genogroups, a comprehensive epitope analysis based on various bioinformatics technology is highly desired for future potential antibody development in clinical diagonosis and treatment. Methods A total of 18 full-length human norovirus capsid protein sequences were downloaded from GenBank. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the protein antigen spatial epitope prediction webserver (SEPPA) for predicting the possible spatial epitopes with the default threshold. The results were processed using the Biosoftware. Results Compared with GI, we found that the GII genogroup had four deletions and two special insertions in the VP1 region. The predicted conformational epitope regions mainly concentrated on N-terminal (1~96), Middle Part (298~305, 355~375) and C-terminal (560~570). We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. Conclusions The predicted conformational epitope regions of norovirus VP1 mainly concentrated on N-terminal, Middle Part and C-terminal. We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. The overlapping with experimental epitopes indicates the important role of latest computational technologies. With the fast development of computational immunology tools, the bioinformatics pipeline will be more and more critical to vaccine design. PMID:23514273

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

    PubMed

    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

  14. Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gp120 helper epitopes1

    PubMed Central

    Li, Hualin; Xu, Chong-Feng; Blais, Steven; Wan, Qi; Zhang, Hui-Tang; Landry, Samuel J.; Hioe, Catarina E.

    2010-01-01

    Glycosylation of HIV-1 envelope gp120 determines not only the proper structure, but also the immune responses against this antigen. While glycans may be part of specific epitopes or shield other epitopes from T cells and antibodies, this study provides evidence for a different immunomodulatory function of glycans associated with gp120 residues N230 and N448. These glycans are required for efficient MHC class II-restricted presentation of nearby CD4 T-cell epitopes, even though they are not part of the epitopes. The glycans do not affect CD4 T cell recognition of more distant epitopes, and are not essential for the proper folding and function of gp120. Data on CD4 T-cell recognition of N448 mutants combined with proteolysis analyses and surface electrostatic potential calculation around residue N448 support the notion that N448-glycan near the epitope's C-terminus renders the site to be surface accessible and allows its efficient processing. In contrast, the N230-glycan contributes to the nearby epitope presentation at a step other than the proteolytic processing of the epitope. Hence, N-glycans can determine CD4 T-cell recognition of nearby gp120 epitopes by regulating the different steps in the MHC class II processing and presentation pathway after APCs acquire the intact gp120 antigen exogenously. Modifications of amino acids bearing glycans at the C termini of gp120 helper epitopes may prove to be a useful strategy for enhancing the immunogenicity of HIV-1 envelope gp120. PMID:19414790

  15. IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

    PubMed Central

    Lee, Mey-Fann; Chang, Chia-Wei; Song, Pei-Pong; Hwang, Guang-Yuh; Lin, Shyh-Jye

    2015-01-01

    Purpose Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. Methods The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. Results Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. Conclusions Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy. PMID:25749772

  16. Epitope prediction algorithms for peptide-based vaccine design.

    PubMed

    Florea, Liliana; Halldórsson, Bjarni; Kohlbacher, Oliver; Schwartz, Russell; Hoffman, Stephen; Istrail, Sorin

    2003-01-01

    Peptide-based vaccines, in which small peptides derived from target proteins (eptiopes) are used to provoke an immune reaction, have attracted considerable attention recently as a potential means both of treating infectious diseases and promoting the destruction of cancerous cells by a patient's own immune system. With the availability of large sequence databases and computers fast enough for rapid processing of large numbers of peptides, computer aided design of peptide-based vaccines has emerged as a promising approach to screening among billions of possible immune-active peptides to find those likely to provoke an immune response to a particular cell type. In this paper, we describe the development of three novel classes of methods for the prediction problem. We present a quadratic programming approach that can be trained on quantitative as well as qualitative data. The second method uses linear programming to counteract the fact that our training data contains mostly positive examples. The third class of methods uses sequence profiles obtained by clustering known epitopes to score candidate peptides. By integrating these methods, using a simple voting heuristic, we achieve improved accuracy over the state of the art. PMID:16826643

  17. Epitope located N-glycans impair the MHC-I epitope generation and presentation.

    PubMed

    Chiritoiu, Gabriela N; Jandus, Camilla; Munteanu, Cristian V A; Ghenea, Simona; Gannon, Philippe O; Romero, Pedro; Petrescu, Stefana M

    2016-06-01

    The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells. PMID:26701645

  18. Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes.

    PubMed

    Upadhyay, Chitra; Ammayappan, Arun; Patel, Deendayal; Kovesdi, Imre; Vakharia, Vikram N

    2011-02-01

    The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector. PMID:21106739

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

    PubMed

    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

  20. Identification of Autoantigen Epitopes in Alopecia Areata.

    PubMed

    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

  1. Guided folding takes a start from the molecular imprinting of structured epitopes.

    PubMed

    Cenci, L; Guella, G; Andreetto, E; Ambrosi, E; Anesi, A; Bossi, A M

    2016-08-25

    A biomimetic route towards assisted folding was explored. Molecularly imprinted polymeric nanoparticles (MIP NPs), i.e. biomimetics with entailed molecular recognition properties made by a template assisted synthesis, were prepared to target a structured epitope: the cystine containing peptide CC9ox, which corresponds to the apical portion of the β-hairpin hormone Hepcidin-25. The structural selection was achieved by the MIP NPs; moreover, the MIP NPs demonstrated favouring the folding of the linear random peptide (CC9red) into the structured one (CC9ox), anticipating the future role of the MIP NPs as in situ nanomachines to counteract folding defects. PMID:27524659

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  6. Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation

    PubMed Central

    Schellens, Ingrid M.; Meiring, Hugo D.; Hoof, Ilka; Spijkers, Sanne N.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Costa, Ana I.; Vennema, Harry; Keşmir, Can; van Baarle, Debbie; van Els, Cécile A. C. M.

    2015-01-01

    Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. ­Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV. PMID:26579122

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

    PubMed

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

    2013-01-01

    Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES. PMID:24358361

  8. Identification of epitopes recognised by mucosal CD4(+) T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7.

    PubMed

    Corbishley, Alexander; Connelley, Timothy K; Wolfson, Eliza B; Ballingall, Keith; Beckett, Amy E; Gally, David L; McNeilly, Tom N

    2016-01-01

    Vaccines targeting enterohaemorrhagic Escherichia coli (EHEC) O157:H7 shedding in cattle are only partially protective. The correlates of protection of these vaccines are unknown, but it is probable that they reduce bacterial adherence at the mucosal surface via the induction of blocking antibodies. Recent studies have indicated a role for cellular immunity in cattle during colonisation, providing an impetus to understand the bacterial epitopes recognised during this response. This study mapped the epitopes of 16 EHEC O157:H7 proteins recognised by rectal lymph node CD4(+) T-cells from calves colonised with Shiga toxin producing EHEC O157:H7 strains. 20 CD4(+) T-cell epitopes specific to E. coli from 7 of the proteins were identified. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4(+) T-cell populations from multiple animals of different major histocompatibility complex class II haplotypes. These T-cell epitopes are missing from many Intimin constructs used in published vaccine trials, but are relatively conserved across a range of EHEC serotypes, offering the potential to develop cross protective vaccines. Antibodies recognising H7 flagellin have been consistently identified in colonised calves; however CD4(+) T-cell epitopes from H7 flagellin were not identified in this study, suggesting that H7 flagellin may act as a T-cell independent antigen. This is the first time that the epitopes recognised by CD4(+) T-cells following colonisation with an attaching and effacing pathogen have been characterised in any species. The findings have implications for the design of antigens used in the next generation of EHEC O157:H7 vaccines. PMID:27590451

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

    SciTech Connect

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

    2014-09-26

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

  10. Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

    SciTech Connect

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

    2012-06-28

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

  11. Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

    PubMed

    Yu, Yongjiao; Fu, Lu; Shi, Yuhua; Guan, Shanshan; Yang, Lan; Gong, Xin; Yin, He; He, Xiaoqiu; Liu, Dongni; Kuai, Ziyu; Shan, Yaming; Wang, Song; Kong, Wei

    2015-12-01

    Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed. PMID:26455781

  12. Cloning and sequencing of the gene for alpha antigen from Mycobacterium avium and mapping of B-cell epitopes.

    PubMed Central

    Ohara, N; Matsuo, K; Yamaguchi, R; Yamazaki, A; Tasaka, H; Yamada, T

    1993-01-01

    The complete nucleotide sequence of alpha antigen secreted from Mycobacterium avium (A-alpha) was determined. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein with a molecular mass of 30,811 Da. This is the first sequence of A-alpha. Comparisons between A-alpha and alpha antigens of Mycobacterium leprae, Mycobacterium bovis BCG, and Mycobacterium kansasii showed highly homologous regions which suggested a conserved functional domain and two less-homologous regions. Serological analysis of recombinant A-alpha, expressed by a series of deletion constructs, indicated the possibility that A-alpha carries at least six B-cell epitopes. The three antigenic determinants were common to Mycobacterium tuberculosis, M. kansasii, and M. avium. The results also suggested the possibility that there are three species-specific epitopes. Images PMID:7681039

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

    PubMed

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

    2013-09-13

    We exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering a multivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The delivery platform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remarkable endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the most abundant influenza internal protein with the conserved primary structure. Two different M1 immunodominant epitopes were separately inserted in Dd external positions without destroying the particles' dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system were properly presented by human dendritic cells triggering efficient activation of antigen-specific T cells responses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens. These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulate cellular immune responses. PMID:23880363

  14. Benchmarking B cell epitope prediction: underperformance of existing methods.

    PubMed

    Blythe, Martin J; Flower, Darren R

    2005-01-01

    Sequence profiling is used routinely to predict the location of B-cell epitopes. In the postgenomic era, the need for reliable epitope prediction is clear. We assessed 484 amino acid propensity scales in combination with ranges of plotting parameters to examine exhaustively the correlation of peaks and epitope location within 50 proteins mapped for polyclonal responses. After examining more than 10(6) combinations, we found that even the best set of scales and parameters performed only marginally better than random. Our results confirm the null hypothesis: Single-scale amino acid propensity profiles cannot be used to predict epitope location reliably. The implication for studies using such methods is obvious. PMID:15576553

  15. Immunogenicity of polysaccharides conjugated to peptides containing T- and B-cell epitopes.

    PubMed Central

    Lett, E; Gangloff, S; Zimmermann, M; Wachsmann, D; Klein, J P

    1994-01-01

    To develop a general model of polysaccharide-peptide vaccine, we have investigated the efficiency of linear peptides derived from protein SR, and adhesin of the I/II protein antigen family of oral streptococci, to act as carriers for two T cell-independent polysaccharides: serogroup f polysaccharide from Streptococcus mutans OMZ 175 (poly f) and Saccharomyces cerevisiae mannan. Peptide 3 (YEKEPTPPTRTPDQ) and peptide 6 (TPEDPTDPTDPQDPSS), accessible on the native SR protein as demonstrated by their reactivity in enzyme-linked immunosorbent assays with rat antisera raised against protein SR, correspond to immunodominant regions of SR. Peptide 3 contains at least one B- and one T-cell epitope, as demonstrated by its ability to induce peptide- and SR-specific antibody responses without any carrier and to stimulate the proliferation of rat lymph node cells primed either with free peptide or native SR, whereas peptide 6 contains only B-cell epitope(s). Peptide 3 was then covalently coupled though reductive amination to either poly f or mannan, and peptide 6 was coupled to poly f. Subcutaneous immunizations of rats with poly f-peptide 3 or mannan-peptide 3 conjugates produced a systemic immunoglobulin M (IgM) and IgG antibody response, and the elicited antibodies reacted with free poly f or mannan, peptide 3, protein SR, and S. mutans or S. cerevisiae whole cells. Rats immunized with poly f-peptide 6 did not develop any antipeptide or anti-SR response. Furthermore, a booster immunization of animals with poly f-peptide 3 or mannan-peptide 3 conjugates induced high titers of anti-peptide 3, anti-poly f, and antimannan antibodies, which occurred quickly. The response is anamnestic for the peptide and the polysaccharides and is characterized by an Ig switch from IgM to IgG. The data presented here confirm that the presence of B- and T-cell epitopes is necessary to induce an anamnestic antipeptide response and that a peptide containing relevant B- and T-cell epitopes can act

  16. Elicitation of structure-specific antibodies by epitope scaffolds

    PubMed Central

    Ofek, Gilad; Guenaga, F. Javier; Schief, William R.; Skinner, Jeff; Baker, David; Wyatt, Richard; Kwong, Peter D.

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant “2F5-epitope scaffolds” possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p < 0.04), while antibody responses against the graft—as dissected residue-by-residue with alanine substitutions—resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation. PMID:20876137

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

    PubMed

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

    2015-01-01

    Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes. PMID:26381649

  18. Characterization of Conformation-dependent Prion Protein Epitopes*

    PubMed Central

    Kang, Hae-Eun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason; Hunter, Nora; Telling, Glenn C.

    2012-01-01

    Whereas prion replication involves structural rearrangement of cellular prion protein (PrPC), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrPSc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain. PMID:22948149

  19. B-cell responses to myelin basic protein and its epitopes in autoimmune encephalomyelitis induced by Semple rabies vaccine.

    PubMed

    Piyasirisilp, S; Hemachudha, T; Griffin, D E

    1999-08-01

    Semple rabies vaccine is composed of rabies virus-infected sheep or goat brain inactivated with phenol and is administered daily after exposure for 14-21 days. Semple rabies vaccine-induced autoimmune encephalomyelitis (SAE) has clinico-pathological findings of demyelination similar to experimental autoimmune encephalomyelitis (EAE) caused by injection of central nervous system tissue or purified myelin proteins into experimental animals and frequently studied as a model for the human demyelinating disease, multiple sclerosis (MS). T-cell-mediated immune responses play a major role in induction of EAE, and antibody responses enhance disease severity. We studied the antibody responses to myelin basic protein (MBP) in 24 Thai patients with SAE and 77 control individuals to define the linear epitopes in human MBP that are encephalitogenic. Antibody levels were assessed by ELISA using native human MBP or synthetic MBP peptides of 20 amino acids. The major B-cell epitope was MBP61-80 and a minor epitope was MBP106-140 in SAE while in MS the major B-cell epitope is MBP84-96. MBP61-80-specific IgG1 and IgG3 levels were significantly higher in patients than controls while IgG2 and IgG4 were not. The data support the hypothesis that autoreactive Th1 cells induce SAE. The difference in B-cell epitope recognition may be due to differences in the genetic backgrounds of the populations studied or may reflect underlying differences in the pathogenesis of SAE and MS. PMID:10430042

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

    PubMed

    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

  1. B-Cell Epitope Mapping of the VapA Protein of Rhodococcus equi: Implications for Early Detection of R. equi Disease in Foals

    PubMed Central

    Vanniasinkam, Thiru; Barton, Mary D.; Heuzenroeder, Michael W.

    2001-01-01

    Linear B-cell epitopes of the Rhodococcus equi virulence-associated protein (VapA) were mapped using a synthetic peptide bank in this study. The peptides were screened in an enzyme-linked immunosorbent assay (ELISA) with a total of 70 sera from foals with current R. equi disease (51 sera), as well as from foals that had either recovered from R. equi infection 10 months previously (3 sera) or that had no known history of R. equi disease (16 sera). An epitope with the sequence NLQKDEPNGRA was identified and was universally recognized by all 51 sera from foals with R. equi disease and was not recognized by any of the other sera. There was poor reactivity between all sera and peptides relating to other areas of the VapA protein. It is proposed that an ELISA based upon a defined peptide epitope may be used in an improved serological diagnostic test for R. equi infection in foals. PMID:11283104

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-19

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

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

    PubMed Central

    Alderete, JF; Neace, Calvin J

    2013-01-01

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

  5. Antibodies Recognizing Mycobacterium avium paratuberculosis Epitopes Cross-React with the Beta-Cell Antigen ZnT8 in Sardinian Type 1 Diabetic Patients

    PubMed Central

    Masala, Speranza; Paccagnini, Daniela; Cossu, Davide; Brezar, Vedran; Pacifico, Adolfo; Ahmed, Niyaz; Mallone, Roberto; Sechi, Leonardo A.

    2011-01-01

    The environmental factors at play in the pathogenesis of type 1 diabetes (T1D) remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP) is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D) and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs) against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8) could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52–65% of T1D patients, but only in 5–7% of T2D and 3–5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals. PMID:22046415

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

    PubMed

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

    2016-04-01

    Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canine-Gp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5-6 IU/mL in mice and 7-9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%-80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies. PMID:27068655

  7. Collections Conservation.

    ERIC Educational Resources Information Center

    DeCandido, Robert

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

  8. Identification of three new type-specific antigen epitopes in the capsid protein of porcine circovirus type 1.

    PubMed

    Huang, Liping; Lu, Yuehua; Wei, Yanwu; Guo, Longjun; Liu, Changming

    2012-07-01

    Porcine circovirus type 1 (PCV1) has been identified as a contaminant of porcine kidney cell line (PK-15). Serological evidence and genetic studies have suggested that PCV1 is widespread in domestic pigs. In this study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated against a recombinant PCV1 Cap protein (PCV1-Cap), which was expressed using the baculovirus system. PEPSCAN analysis was used to identify epitopes on the PCV1-Cap with mAbs and pAbs. Three linear B-cell epitopes, including residues (85)GGTNPLP(91), (162)FTPKPELDKTIDWFHPNNK(180) and (219)YVQFREFILKDPLNK(233), specific for PCV1-Cap, were finely defined. These results will facilitate future investigations into antigenic differences and differential diagnosis between PCV1 and PCV2. PMID:22437253

  9. T Cell Epitopes and Post-Translationally Modified Epitopes in Type 1 Diabetes

    PubMed Central

    McGinty, John W.; Marré, Meghan L.; Bajzik, Veronique; Piganelli, Jon D.; James, Eddie A.

    2016-01-01

    Type 1 diabetes (T1D) is an autoimmune disease in which progressive loss of self-tolerance, evidenced by accumulation of auto-antibodies and auto-reactive T cells that recognize diverse self-proteins, leads to immune-mediated destruction of pancreatic beta cells and loss of insulin secretion. In this review, we discuss antigens and epitopes in T1D and the role that post-translational modifications play in circumventing tolerance mechanisms and increasing antigenic diversity. Emerging data suggest that, analogous to other autoimmune diseases such as rheumatoid arthritis and celiac disease, enzymatically modified epitopes are preferentially recognized in T1D. Modifying enzymes such as peptidyl deiminases and tissue transglutaminase are activated in response to beta cell stress, providing a mechanistic link between post-translational modification and interactions with the environment. Although studies of such responses in the at-risk population have been limited, current data suggests that breakdown in tolerance through post-translational modification represents an important checkpoint in the development of T1D. PMID:26370701

  10. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR. PMID:7594584

  11. Ex vivo detection of adenovirus specific CD4{sup +} T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T{sub HELPER} cells following stem cell transplantation

    SciTech Connect

    Serangeli, Celine; Bicanic, Oliver; Scheible, Michael H.; Lang, Peter; Handgretinger, Rupert

    2010-02-20

    Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4{sup +} T-cell responses against the Hexon-protein, but the frequency of specific T{sub HELPER} cells is extremely low or not detectable ex vivo and preference for different CD4{sup +} T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4{sup +}-responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4{sup +}-proliferation in >50% of individuals, confirmed by intracellular IFN-gamma detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4{sup +} T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4{sup +} T cells for adoptive T-cell transfer against HAdV-infection post SCT.

  12. Identification of Novel HLA-A2-Restricted Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Epitopes Predicted by the HLA-A2 Supertype Peptide-Binding Motif

    PubMed Central

    Altfeld, Marcus A.; Livingston, Brian; Reshamwala, Neha; Nguyen, Phuong T.; Addo, Marylyn M.; Shea, Amy; Newman, Mark; Fikes, John; Sidney, John; Wentworth, Peggy; Chesnut, Robert; Eldridge, Robert L.; Rosenberg, Eric S.; Robbins, Gregory K.; Brander, Christian; Sax, Paul E.; Boswell, Steve; Flynn, Theresa; Buchbinder, Susan; Goulder, Philip J. R.; Walker, Bruce D.; Sette, Alessandro; Kalams, Spyros A.

    2001-01-01

    Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines. PMID:11152503

  13. Fragment-based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping

    PubMed Central

    Begley, Darren W.; Zheng, Suxin; Varani, Gabriele

    2010-01-01

    Solution state nuclear magnetic resonance (NMR1) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the co-factor binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations validated using experimentally-derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor binding site. PMID:20626411

  14. The Calmodulin-Binding, Short Linear Motif, NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

    PubMed Central

    Taiakina, Valentina; Boone, Adrienne N.; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca2+ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca2+-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. PMID:23626724

  15. The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

    PubMed

    Taiakina, Valentina; Boone, Adrienne N; Fux, Julia; Senatore, Adriano; Weber-Adrian, Danielle; Guillemette, J Guy; Spafford, J David

    2013-01-01

    NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+) concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+)-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels. PMID:23626724

  16. Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium.

    PubMed

    Wang, J; Michel, V; Leclerc, C; Hofnung, M; Charbit, A

    1999-01-01

    We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites of a carrier protein. By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number of different foreign epitopes at the bacterial surface. In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.e.--the C3 epitope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145)--at the tip of the most distal and largest surface exposed region of LamB (after residues 386, into loop L9). We also used two previously constructed LamB hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two LamB proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site). The LamB hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S. typhimurium, SL3261. In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained. For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153. Moreover, the LamB hybrid corresponding to the simultaneous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hepatitis B antibodies. PMID:10078601

  17. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

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

    PubMed

    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

  19. Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens.

    PubMed

    Wang, Jian; Lin, Yahui; Cai, Pengfei; Wang, Heng

    2011-01-01

    The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines. PMID

  20. Epitope specificity of anti‐HA2 antibodies induced in humans during influenza infection

    PubMed Central

    Staneková, Zuzana; Mucha, Vojtech; Sládková, Tatiana; Blaškovičová, Hana; Kostolanský, František; Varečková, Eva

    2012-01-01

    Please cite this paper as: Stanekováet al. (2012) Epitope specificity of anti‐HA2 antibodies induced in humans during influenza infection. Influenza and Other Respiratory Viruses 6(6), 389–395. Background  The conserved, fusion‐active HA2 glycopolypeptide (HA2) subunit of influenza A hemagglutinin comprises four distinct antigenic sites. Monoclonal antibodies (MAbs) recognizing three of these sites are broadly cross‐reactive and protective. Objectives  This study aimed to establish whether antibodies specific to these three antigenic sites were elicited during a natural influenza infection or by vaccination of humans. Methods  Forty‐five paired acute and convalescent sera from individuals with a confirmed influenza A (subtype H3) infection were examined for the presence of HA2‐specific antibodies. The fraction of antibodies specific to three particular antigenic sites (designated IIF4, FC12, and CF2 here) was investigated using competitive enzyme immunoassay. Results  Increased levels of antibodies specific to an ectodomain of HA2 (EHA2: N‐terminal residues 23–185 of HA2) were detected in 73% of tested convalescent sera (33/45), while an increased level of antibodies specific to the HA2 fusion peptide (N‐terminal residues 1–38) was induced in just 15/45 individuals (33%). Competitive assays confirmed that antibodies specific to the IIF4 epitope (within HA2 residues 125–175) prevailed in 86% (13/15) over those specific to the other two epitopes during infection. However, only a negligible increase in HA2‐specific antibodies was detectable following vaccination with a current subunit vaccine. Conclusions  We observed that the antigenic site localized within N‐terminal HA2 residues 125–175 was more immunogenic than that within residues 1–38 (HA2 fusion protein), although both are weak natural immunogens. We suggest that new anti‐influenza vaccines should include HA2 (or specific epitopes localized within this

  1. T-cell epitopes in Pf155/RESA, a major candidate for a Plasmodium falciparum malaria vaccine.

    PubMed Central

    Kabilan, L; Troye-Blomberg, M; Perlmann, H; Andersson, G; Högh, B; Petersen, E; Björkman, A; Perlmann, P

    1988-01-01

    Immunogens included in a subunit vaccine should contain both B- and T-cell-activating sites to ensure anamnestic responses following reinfection after vaccination as well as antibody-independent cellular immunity. The Plasmodium falciparum antigen Pf155/RESA, a major candidate for a vaccine against the asexual blood stages of this malaria parasite, was investigated for T-cell epitopes in its C-terminal amino acid repeat region, a region known to be conserved in different P. falciparum strains. It was found to contain several related sequences that activated T cells from humans primed to P. falciparum by natural exposure, to proliferation, and/or interferon-gamma release in vitro. T cells from approximately half of the donor group investigated responded to the intact protein, and 65% of these responders also responded to short synthetic peptides, probably representing a small number of partly overlapping T-cell epitopes. Thus, sequences from the C terminus of Pf155 may be suitable constituents of a P. falciparum subunit vaccine and also provide a basis for epitope-specific epidemiological studies relating cellular immune responses in vitro to clinical immunity and P. falciparum endemicity. PMID:2456577

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

    PubMed Central

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

    2015-01-01

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

  3. Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva.

    PubMed

    Obara, Isaiah; Ulrike, Seitzer; Musoke, Tony; Spooner, Paul R; Jabbar, Ahmed; Odongo, David; Kemp, Stephen; Silva, Joana C; Bishop, Richard P

    2015-05-01

    Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo. PMID:25673078

  4. A sequence pattern common to T cell epitopes.

    PubMed Central

    Rothbard, J B; Taylor, W R

    1988-01-01

    An analysis of the known cytotoxic and helper T cell epitopes has revealed similarity within their primary sequences. These similar motifs, characteristic of the known determinants, have been incorporated into predictive templates that have been used successfully to define eight helper and three cytotoxic epitopes in four different proteins. When the defined epitopes are segregated by restriction element, allele specific subpatterns emerge centering around the general pattern. The presence of similarities argues that the binding of peptide antigens to class I and class II is similar in nature. In addition, these motifs can be used to predict accurately areas within proteins capable of being recognized by individual MHC class I and class II molecules. PMID:2452085

  5. Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis†

    PubMed Central

    Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

    2005-01-01

    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations. PMID:15795253

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  7. Identification and Characterization of an Immunogenic Hybrid Epitope Formed by both HIV gp120 and Human CD4 Proteins ▿

    PubMed Central

    Lewis, George K.; Fouts, Timothy R.; Ibrahim, Sani; Taylor, Brian M.; Salkar, Rachita; Guan, Yongjun; Kamin-Lewis, Roberta; DeVico, Anthony L.

    2011-01-01

    Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection. PMID:21994452

  8. Linear Accelerators

    NASA Astrophysics Data System (ADS)

    Sidorin, Anatoly

    2010-01-01

    In linear accelerators the particles are accelerated by either electrostatic fields or oscillating Radio Frequency (RF) fields. Accordingly the linear accelerators are divided in three large groups: electrostatic, induction and RF accelerators. Overview of the different types of accelerators is given. Stability of longitudinal and transverse motion in the RF linear accelerators is briefly discussed. The methods of beam focusing in linacs are described.

  9. Linear Accelerators

    SciTech Connect

    Sidorin, Anatoly

    2010-01-05

    In linear accelerators the particles are accelerated by either electrostatic fields or oscillating Radio Frequency (RF) fields. Accordingly the linear accelerators are divided in three large groups: electrostatic, induction and RF accelerators. Overview of the different types of accelerators is given. Stability of longitudinal and transverse motion in the RF linear accelerators is briefly discussed. The methods of beam focusing in linacs are described.

  10. Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof

    DOEpatents

    Stupp, Samuel I.; Guler, Mustafa O.

    2008-11-18

    Branched peptide amphiphilic compounds incorporating one or residues providing a pendant amino group for coupling one or more epitope sequences thereto, such compounds and related compositions for enhanced epitope presentation.

  11. Variable sequences in a mosaic-like domain of meningococcal tbp2 encode immunoreactive epitopes.

    PubMed

    Rokbi, B; Maitre-Wilmotte, G; Mazarin, V; Fourrichon, L; Lissolo, L; Quentin-Millet, M J

    1995-10-15

    Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure. PMID:7590185

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

    PubMed

    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

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

    PubMed

    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

  14. Thermo-sensitive imprinted polymer embedded carbon dots using epitope approach.

    PubMed

    Li, Dong-Yan; Zhang, Xue-Mei; Yan, Yun-Jing; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui

    2016-05-15

    A new type of thermo-sensitive receptor carbon dots/SiO2/molecularly imprinted polymer (CDs/SiO2/MIP) was prepared by surface imprinting procedure and the epitope approach. The synthetic CDs/SiO2/MIP was able to selectively capture target protein with fluorescence quenching via the special interaction between them and the recognition cavities. The receptor exhibited the linear fluorescence quenching to cytochrome c (cyt c) in the range of 0.1-40 μM, and the detection limit was 89 nM. The precision for five replicate detection of cyt c at 20 μM was 3.11%. Moreover, the receptor owned the temperature-sensitive element that allowed for swelling and shrinking in response to temperature changes to realize recognition of the target cytochrome c. The proposed strategy revealed the feasibility of fabrication of a thermo-sensitive imprinted polymer based on CDs and surface imprinting procedure and the epitope approach. PMID:26706940

  15. Identification of a highly immunoreactive epitope of Brugia malayi TPx recognized by the endemic sera.

    PubMed

    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 < 0.05) against the sera of putatively immune individuals compared to the nonendemic control sera. It also showed high reactivity against the sera of patients with chronic pathology and 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

  16. Multimerized T cell epitopes protect from experimental autoimmune diabetes by inducing dominant tolerance.

    PubMed

    Piaggio, Eliane; Mars, Lennart T; Cassan, Cécile; Cabarrocas, Julie; Hofstätter, Maria; Desbois, Sabine; Bergereau, Emilie; Rötzschke, Olaf; Falk, Kirsten; Liblau, Roland S

    2007-05-29

    Immunotherapy by using multimerized self-peptides has demonstrated a clear protective effect on experimental models of autoimmune diseases. However, the mechanisms involved remain ill-defined. Here we have evaluated the therapeutic efficacy of multimerized self-peptides at the effector phase of autoimmune diabetes and examined their mechanisms of action. Diabetes was induced in rat insulin promoter-hemagglutinin (HA) mice expressing HA in pancreatic beta-cells by adoptive transfer of HA(110-119)-specific T helper 1 cells. Complete protection was provided by low doses of the HA 4-mer consisting of four covalently linked linear HA(107-119) peptides. In vivo, the 4-mer appeared to act directly on the pathogenic HA-specific T helper 1 cells and indirectly by activation/recruitment of lymphocytes with regulatory properties so that mice became resistant to a second transfer of diabetogenic T cells. This effect was associated with a recruitment of Foxp3(+) CD4 T cells around islets. Moreover, we show that dominant protection from autoimmunity was transferable by spleen cells, and that development of this regulatory population was crucially dependent on the lymphocytes from treated rat insulin promoter-HA mice. Thus, immunotherapy using multimerized epitopes emerges as a promising strategy in view of the current identification of self-epitopes that are major targets of the pathogenic CD4 T cell response in autoimmune diseases. PMID:17517665

  17. Multimerized T cell epitopes protect from experimental autoimmune diabetes by inducing dominant tolerance

    PubMed Central

    Piaggio, Eliane; Mars, Lennart T.; Cassan, Cécile; Cabarrocas, Julie; Hofstätter, Maria; Desbois, Sabine; Bergereau, Emilie; Rötzschke, Olaf; Falk, Kirsten; Liblau, Roland S.

    2007-01-01

    Immunotherapy by using multimerized self-peptides has demonstrated a clear protective effect on experimental models of autoimmune diseases. However, the mechanisms involved remain ill-defined. Here we have evaluated the therapeutic efficacy of multimerized self-peptides at the effector phase of autoimmune diabetes and examined their mechanisms of action. Diabetes was induced in rat insulin promoter-hemagglutinin (HA) mice expressing HA in pancreatic β-cells by adoptive transfer of HA110–119-specific T helper 1 cells. Complete protection was provided by low doses of the HA 4-mer consisting of four covalently linked linear HA107–119 peptides. In vivo, the 4-mer appeared to act directly on the pathogenic HA-specific T helper 1 cells and indirectly by activation/recruitment of lymphocytes with regulatory properties so that mice became resistant to a second transfer of diabetogenic T cells. This effect was associated with a recruitment of Foxp3+ CD4 T cells around islets. Moreover, we show that dominant protection from autoimmunity was transferable by spleen cells, and that development of this regulatory population was crucially dependent on the lymphocytes from treated rat insulin promoter-HA mice. Thus, immunotherapy using multimerized epitopes emerges as a promising strategy in view of the current identification of self-epitopes that are major targets of the pathogenic CD4 T cell response in autoimmune diseases. PMID:17517665

  18. Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

    PubMed

    Li, R; Liu, T; Wong, B S; Pan, T; Morillas, M; Swietnicki, W; O'Rourke, K; Gambetti, P; Surewicz, W K; Sy, M S

    2000-08-18

    We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein. PMID:10966770

  19. Data on the epitope mapping of soybean A2 and A3 glycinin.

    PubMed

    Saeed, Hanaa; Gagnon, Christine; Cober, Elroy; Gleddie, Steve

    2016-09-01

    The data information provided in this article relate to our research article "Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts" (Saeed et al., 2016) [1]. Here we provide western blot detection of glycinin subunits by soy-sensitive human sera, ELISA screens with overlapping synthetic peptides (epitope mapping), and various database/server epitope searches. PMID:27294180

  20. Meta-analysis of All Immune Epitope Data in the Flavivirus Genus: Inventory of Current Immune Epitope Data Status in the Context of Virus Immunity and Immunopathology

    PubMed Central

    Greenbaum, Jason; Blythe, Martin; Peters, Bjoern; Sette, Alessandro

    2010-01-01

    Abstract A meta-analysis was performed in order to inventory the immune epitope data related to viruses in the genus Flavivirus. Nearly 2000 epitopes were captured from over 130 individual Flavivirus-related references identified from PubMed and reported as of September 2009. This report includes all epitope structures and associated immune reactivity from the past and current literature, including: the epitope distribution among pathogens and related strains, the epitope distribution among different pathogen antigens, the number of epitopes defined in human and animal models of disease, the relationship between epitopes identified in different disease states following natural (or experimental) infection, and data from studies focused on candidate vaccines. We found that the majority of epitopes were defined for dengue virus (DENV) and West Nile virus (WNV). The prominence of DENV and WNV data in the epitope literature is likely a reflection of their overall worldwide impact on human disease, and the lack of vaccines. Conversely, the relatively smaller number of epitopes defined for the other viruses within the genus (yellow fever and Japanese encephalitis virus) most likely reflects the presence of established prophylaxis and/or their more modest impact on morbidity and mortality globally. Through this work we hope to provide useful data to those working in the area of Flavivirus research. PMID:20565291

  1. Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

    PubMed Central

    Huang, Zhen-Yu; Cairns, Tina M.; Gallagher, John R.; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B.; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2

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

    PubMed Central

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

    2008-01-01

    Background The prediction of conformational B-cell epitopes is one of the most important goals in immunoinformatics. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues of interaction between an antigen and an antibody. Consequently, this area of research has received considerable attention from immunologists, structural biologists and computational biologists. Phage-displayed random peptide libraries are powerful tools used to obtain mimotopes that are selected by binding to a given monoclonal antibody (mAb) in a similar way to the native epitope. These mimotopes can be considered as functional epitope mimics. Mimotope analysis based methods can predict not only linear but also conformational epitopes and this has been the focus of much research in recent years. Though some algorithms based on mimotope analysis have been proposed, the precise localization of the interaction site mimicked by the mimotopes is still a challenging task. Results In this study, we propose a method for B-cell epitope prediction based on mimotope analysis called Pep-3D-Search. Given the 3D structure of an antigen and a set of mimotopes (or a motif sequence derived from the set of mimotopes), Pep-3D-Search can be used in two modes: mimotope or motif. To evaluate the performance of Pep-3D-Search to predict epitopes from a set of mimotopes, 10 epitopes defined by crystallography were compared with the predicted results from a Pep-3D-Search: the average Matthews correlation oefficient (MCC), sensitivity and precision were 0.1758, 0.3642 and 0.6948. Compared with other available prediction algorithms, Pep-3D-Search showed comparable MCC, specificity and precision, and could provide novel, rational results. To verify the capability of Pep-3D-Search to align a motif sequence to a 3D structure for predicting epitopes, 6 test cases were used. The predictive performance of Pep-3D-Search was demonstrated to be superior to that of other

  3. Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Cappelletti, Francesca; Clementi, Massimo; Burioni, Roberto

    2014-01-01

    The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results. PMID:24515481

  4. Linearly Adjustable International Portfolios

    SciTech Connect

    Fonseca, R. J.; Kuhn, D.; Rustem, B.

    2010-09-30

    We present an approach to multi-stage international portfolio optimization based on the imposition of a linear structure on the recourse decisions. Multiperiod decision problems are traditionally formulated as stochastic programs. Scenario tree based solutions however can become intractable as the number of stages increases. By restricting the space of decision policies to linear rules, we obtain a conservative tractable approximation to the original problem. Local asset prices and foreign exchange rates are modelled separately, which allows for a direct measure of their impact on the final portfolio value.

  5. Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

    PubMed Central

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-01-01

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6,564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research. PMID:25445329

  6. Humanization of predicted T-cell epitopes reduces the immunogenicity of chimeric antibodies: new evidence supporting a simple method.

    PubMed

    Roque-Navarro, Lourdes; Mateo, Cristina; Lombardero, Josefa; Mustelier, Geraudis; Fernández, Alicia; Sosa, Katya; Morrison, Sherrie L; Pérez, Rolando

    2003-08-01

    Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between re-shaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential. PMID:14511570

  7. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  8. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGESBeta

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  9. 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.

    PubMed

    Boonsathorn, Naphatsawan; Panthong, Sumolrat; Koksunan, Sarawut; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Yasugi, Mayo; Ono, Ken-Ichiro; Arai, Yasuha; Kurosu, Takeshi; Sawanpanyalert, Pathom; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-09-26

    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. PMID:25204499

  10. On integrable conservation laws

    PubMed Central

    Arsie, Alessandro; Lorenzoni, Paolo; Moro, Antonio

    2015-01-01

    We study normal forms of scalar integrable dispersive (not necessarily Hamiltonian) conservation laws, via the Dubrovin–Zhang perturbative scheme. Our computations support the conjecture that such normal forms are parametrized by infinitely many arbitrary functions that can be identified with the coefficients of the quasi-linear part of the equation. Moreover, in general, we conjecture that two scalar integrable evolutionary partial differential equations having the same quasi-linear part are Miura equivalent. This conjecture is also consistent with the tensorial behaviour of these coefficients under general Miura transformations. PMID:25568614

  11. Identification of a cross-reactive continuous B-cell epitope in enterotoxigenic Escherichia coli colonization factor antigen I.

    PubMed Central

    Rudin, A; Svennerholm, A M

    1996-01-01

    Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine by means of several antigenically distinct colonization factors (CFs). Several of these CFs have very significant amino acid sequence similarity or identity, particularly in the N-terminal end. We have previously shown that a monoclonal antibody (MAb) raised against the subunits of colonization factor antigen I (CFA/I) fimbriae, which reacts with a peptide corresponding to the 25 N-terminal amino acids of such subunits, can inhibit attachment to intestinal cells of ETEC expressing heterologous as well as homologous CFs, with related amino acid sequences. In this study we have, by means of Pepscan analysis, determined the sequence of the MAb-specific linear epitope to be 15IDLLQ19. Parenteral immunization of rabbits with an N-terminal 25-mer synthetic peptide of CFA/I fimbrial subunit, either covalently coupled to bovine serum albumin or uncoupled, induced high titers of specific antibodies against this peptide as well as against CFA/I fimbriae. Increased titers against several heterologous CF fimbriae with a related N-terminal sequence were also induced, whereas no increase was seen against fimbriae with an unrelated sequence. Neither antisera against the coupled peptide nor antisera against the uncoupled peptide inhibited binding of CF-expressing bacteria to the human intestinal cell line Caco-2 in spite of high titers. The difference in the inhibitory capabilities of the antipeptide sera and the MAb might be due to slightly different epitope specificities. Thus, whereas the antipeptide sera bound to several continuous epitopes in the N-terminal end, none of them reacted specifically with the epitope 15IDLLQ19. PMID:8890199

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

    PubMed

    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

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

    SciTech Connect

    Korber, Bette; Szinger, James

    2009-01-01

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

  14. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

    PubMed Central

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

  15. Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

    PubMed

    Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

    2015-01-01

    Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

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

    PubMed

    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

  17. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  18. Altered response hierarchy and increased T-cell breadth upon HIV-1 conserved element DNA vaccination in macaques.

    PubMed

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K; Jalah, Rashmi; Broderick, Kate E; Sardesai, Niranjan Y; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag) elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag) increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  19. Conservation Presentation.

    ERIC Educational Resources Information Center

    Friday, Gerald

    2001-01-01

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

  20. Expression of antigenic epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) in a modified live-attenuated porcine circovirus type 2 (PCV2) vaccine virus (PCV1-2a) as a potential bivalent vaccine against both PCV2 and PRRSV.

    PubMed

    Piñeyro, Pablo E; Kenney, Scott P; Giménez-Lirola, Luis G; Heffron, C Lynn; Matzinger, Shannon R; Opriessnig, Tanja; Meng, Xiang-Jin

    2015-12-01

    Co-infection of pigs in the field with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is common and poses a major concern in effective control of PCV2 and PRRSV. We previously demonstrated that insertion of foreign epitope tags in the C-terminus of PCV2 ORF2 produced infectious virions that elicited humoral immune responses against both PCV2 capsid and inserted epitope tags. In this study, we aimed to determine whether the non-pathogenic chimeric virus PCV1-2a, which is the basis for the licensed PCV2 vaccine Fostera PCV, can express PRRSV antigenic epitopes, thus generating dual immunity as a potential bivalent vaccine against both PCV2 and PPRSV. Four different linear B-cell antigenic epitopes of PRRSV were inserted into the C-terminus of the capsid gene of the PCV1-2a vaccine virus. We showed that insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not impair the replication of the resulting PCV1-2a-PRRSVEPI chimeric viruses in vitro. The four chimeric PCV1-2a viruses expressing PRRSV B-cell linear epitopes were successfully rescued and characterized. An immunogenicity study in pigs revealed that two of the four chimeric viruses, PCV1-2a-PRRSVEPIGP3IG and PCV1-2a-PRRSVEPIEPIGP5IV, elicited neutralizing antibodies against PRRSV VR2385 as well as PCV2 (strains PCV2a, PCV2b, and mPCV2b). The results have important implications for exploring the potential use of PCV1-2a vaccine virus as a live virus vector to develop bivalent MLVs against both PCV2 and PRRSV. PMID:26239318

  1. Linear Collisions

    ERIC Educational Resources Information Center

    Walkiewicz, T. A.; Newby, N. D., Jr.

    1972-01-01

    A discussion of linear collisions between two or three objects is related to a junior-level course in analytical mechanics. The theoretical discussion uses a geometrical approach that treats elastic and inelastic collisions from a unified point of view. Experiments with a linear air track are described. (Author/TS)

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

    Cancer.gov

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. HLA-A2 and B35 Restricted Hantaan Virus Nucleoprotein CD8+ T-Cell Epitope-Specific Immune Response Correlates with Milder Disease in Hemorrhagic Fever with Renal Syndrome

    PubMed Central

    Yuan, Bin; Wang, Meiliang; Zhang, Yun; Xu, Zhuwei; Zhang, Chunmei; Zhang, Yusi; Liu, Bei; Yi, Jing; Yang, Kun; Yang, Angang; Zhuang, Ran; Jin, Boquan

    2013-01-01

    Background Hantaan virus (HTNV) infection in humans is a serious public health concern in Asia. A potent T cell activation peptide vaccine from HTNV structure protein represents a promising immunotherapy for disease control. However, the T cell epitopes of the HTNV restricted by the HLA alleles and the role of epitope-specific T cell response after HTNV infection remain largely unexplored. Methodology/Principal Findings Five well-conserved novel CD8+ T-cell epitopes of the HTNV nucleoprotein restricted by the most popular HLA alleles in Chinese Han population were defined with interferon-γ enzyme-linked immunospot assay in 37 patients infected with HTNV during hospitalization. Two epitopes aa129–aa137 and aa131–aa139 restricted by HLA-A2 and B35, respectively, were selected to evaluate the epitope-specific CD8+ T-cell response. HLA-peptide pentamer complex staining showed that the frequency of single epitope-specific CD8+ T cell could be detected in patients (95% confidence interval for aa129–aa137: 0.080%–0.208%; for aa131–aa139: 0.030%–0.094%). The frequency of epitope-specific pentamer+ CD8+ T-cell response was much higher in mild/moderate patients than in severe/critical ones at the acute stage of the disease. Moreover, the frequency of epitope-specific CD8+ T cells at acute stage was inversely associated with the peak level of serum creatinine and was positively associated with the nadir platelet counts during the hospitalization. The intracellular cytokine staining and the proliferation assay showed that the effective epitope-specific CD8+ T cells were characterized with the production of interferon-γ, expression of CD69 and the strong capacity of proliferation. Conclusion/Significance The novel HLA class I restricted HTNV nucleoprotein epitopes-specific CD8+ T-cell responses would be closely related with the progression and the severity of the disease, which could provide the first step toward effective peptide vaccine development against HTNV

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

    PubMed

    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

  7. B-Cell Epitopes in NTS-DBL1α of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

    PubMed Central

    Albrecht, Letusa; Blomqvist, Karin; Valentini, Davide; D'Alexandri, Fabio Luiz; Maurer, Markus; Wahlgren, Mats

    2014-01-01

    Plasmodium falciparum is the most lethal of the human malaria parasites. The virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1α-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1α of IT4var60) expressed by parasite FCR3S1.2. The plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1α domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. The other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. In conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1α of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. The data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies. PMID:25438249

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

    PubMed

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

    2016-05-01

    CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion. PMID:26909514

  9. Conservation and Variability of West Nile Virus Proteins

    PubMed Central

    Jung, Keun-Ok; Ramdas, Shweta; Miotto, Olivo; Tan, Tin Wee; Brusic, Vladimir; Salmon, Jerome; August, J. Thomas

    2009-01-01

    West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of ≤1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (≤10% of the WNV sequences analyzed). Eighty-eight fragments of length 9–29 amino acids, representing ∼34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for

  10. LINEAR ACCELERATOR

    DOEpatents

    Christofilos, N.C.; Polk, I.J.

    1959-02-17

    Improvements in linear particle accelerators are described. A drift tube system for a linear ion accelerator reduces gap capacity between adjacent drift tube ends. This is accomplished by reducing the ratio of the diameter of the drift tube to the diameter of the resonant cavity. Concentration of magnetic field intensity at the longitudinal midpoint of the external sunface of each drift tube is reduced by increasing the external drift tube diameter at the longitudinal center region.

  11. Identification of broadly recognized, T helper 1 lymphocyte epitopes in an equine lentivirus

    PubMed Central

    Fraser, Darrilyn G; Oaks, J Lindsay; Brown, Wendy C; McGuire, Travis C

    2002-01-01

    Equine infectious anaemia virus (EIAV) is a horse lentivirus causing lifelong, persistent infection. During acute infection, CD8+ cytotoxic T lymphocytes (CTL) are probably involved in terminating plasma viraemia. However, only a few EIAV CTL epitopes, restricted to fewer horse major histocompatibility complex (MHC) class I alleles, are known. As interferon-γ (IFN-γ)-secreting CD4+, T helper 1 (Th1) lymphocytes promote CTL activity and help maintain memory CTL, identifying broadly recognized EIAV Th1 epitopes would contribute significantly to vaccine strategies seeking to promote strong CTL responses among horses with varying class I haplotypes. To this end, peripheral blood mononuclear cells (PBMC) from 10 MHC disparate, EIAV-infected horses were tested in T-lymphocyte proliferation assays for recognition of peptides from the Gag p26 capsid region and a portion of Pol. Both regions are highly conserved among EIAV isolates, and this Pol region is 51–63% homologueous to other lentiviral Pol proteins. Seven of 10 horses recognized peptide Gag 221–245, and peptides Gag 242–261 and Pol 323–344 were recognized by five and four horses, respectively. Furthermore, the Gag peptides were recognized by two additional horses after resolving their initial plasma viraemia, indicating that these two peptides can be immunodominant early in infection. Gag peptide-responsive PBMC produced only IFN-γ, indicating a Th1 response, while Pol 323–344-responsive PBMC produced IFN-γ both with and without interleukin-4. PBMC from uninfected horses failed to either proliferate or secrete cytokines in response to peptide stimulation. Finally, CD4+ T lymphocytes were required for proliferation responses, as shown by assays using CD4- versus CD8-depleted PBMC. PMID:11918691

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

    SciTech Connect

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

    2009-11-23

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

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

    USGS Publications Warehouse

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

    1996-01-01

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

  14. Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

    PubMed Central

    Reguzova, Alena; Antonets, Denis; Karpenko, Larisa; Ilyichev, Alexander; Maksyutov, Rinat; Bazhan, Sergei

    2015-01-01

    A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine. PMID:25786238

  15. Virus-epitope vaccine design: informatic matching the HLA-I polymorphism to the virus genome.

    PubMed

    Vider-Shalit, Tal; Raffaeli, Shai; Louzoun, Yoram

    2007-02-01

    Attempts to develop peptide vaccines, based on a limited number of peptides face two problems: HLA polymorphism and the high mutation rate of viral epitopes. We have developed a new genomic method that ensures maximal coverage and thus maximal applicability of the peptide vaccine. The same method also promises a large number of epitopes per HLA to prevent escape via mutations. Our design can be applied swiftly in order to face rapidly emerging viral diseases. We use a genomic scan of all candidate peptides and join them optimally. For a given virus, we use algorithms computing: peptide cleavage probability, transfer through TAP and MHC binding for a large number of HLA alleles. The resulting peptide libraries are pruned for peptides that are not conserved or are too similar to self peptides. We then use a genetic algorithm to produce an optimal protein composed of peptides from this list properly ordered for cleavage. The selected peptides represent an optimal combination to cover all HLA alleles and all viral proteins. We have applied this method to HCV and found that some HCV proteins (mainly envelope proteins) represent much less peptide than expected. A more detailed analysis of the peptide variability shows a balance between the attempts of the immune system to detect less mutating peptides, and the attempts of viruses to mutate peptides and avoid detection by the immune system. In order to show the applicability of our method, we have further used it on HIV-I, Influenza H3N2 and the Avian Flu Viruses. PMID:16930710

  16. Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies.

    PubMed

    Bencina, D; Kleven, S H; Elfaki, M G; Snoj, A; Dovc, P; Dorrer, D; Russ, I

    1994-03-01

    Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response. PMID:18671069

  17. The evolutionary conservation of DNA polymerase. alpha

    SciTech Connect

    Miller, M.A.; Korn, D.; Wang, T.S.F. )

    1988-08-25

    The evolutionary conservation of DNA polymerase {alpha} was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase {alpha} monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase {alpha} catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

  18. Identification of a major continuous epitope of human alpha crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

  19. Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

    PubMed Central

    Fong, Rachel H.; Banik, Soma S. R.; Mattia, Kimberly; Barnes, Trevor; Tucker, David; Liss, Nathan; Lu, Kai; Selvarajah, Suganya; Srinivasan, Surabhi; Mabila, Manu; Miller, Adam; Muench, Marcus O.; Michault, Alain; Rucker, Joseph B.; Paes, Cheryl; Simmons, Graham; Kahle, Kristen M.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected

  20. Marketing Conservation.

    ERIC Educational Resources Information Center

    Ellis, William B.

    1987-01-01

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

  1. Colorful Conservation

    ERIC Educational Resources Information Center

    Skophammer, Karen

    2011-01-01

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

  2. [Conservation Units.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

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

  3. [Conservation Units.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin.

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

  4. The developmental regulation of the L2/HNK-1 and L3 carbohydrate epitopes in mouse brain. Evidence for separate control of lipid- and protein-bound epitopes.

    PubMed

    Breen, K C

    1989-04-10

    The carbohydrate epitopes L2/HNK-1 and L3 have previously been identified on various neural cell adhesion molecules and have been suggested to play a role in the mediation of cell-cell adhesion. In this study, the developmental expression of the two epitopes in soluble, membrane-bound and chloroform/methanol-extracted fractions of the constituent mouse brain regions was examined by enzyme-linked immunosorbent assay (ELISA). The protein-bound epitopes were shown to be uniformly developmentally regulated, with levels peaking at postnatal day 20 (P20). The epitopes in a crude chloroform/methanol fraction, however, demonstrated a different pattern, with L2 peaking earlier at postnatal day zero (P0). These results suggest a possible interaction between the control of the two pools of the epitope. PMID:2468531

  5. A General Method to Discover Epitopes from Sera

    PubMed Central

    Whittemore, Kurt; Johnston, Stephen Albert; Sykes, Kathryn; Shen, Luhui

    2016-01-01

    Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen

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

    EPA Science Inventory

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

  7. Common food allergens and their IgE-binding epitopes.

    PubMed

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut. PMID:26433529

  8. Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

    PubMed Central

    Tissot, Alain C.; Renhofa, Regina; Schmitz, Nicole; Cielens, Indulis; Meijerink, Edwin; Ose, Velta; Jennings, Gary T.; Saudan, Philippe; Pumpens, Paul; Bachmann, Martin F.

    2010-01-01

    Background Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. Methodology/Principal Findings Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. Conclusions/Significance AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection. PMID:20352110

  9. Mapping epitopes and antigenicity by site-directed masking

    NASA Astrophysics Data System (ADS)

    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

  10. Synthetic peptide-targeted selection of phage display mimotopes highlights immunogenic features of α-helical vs non-helical epitopes of Taenia solium paramyosin: implications for parasite- and host-protective roles of the protein.

    PubMed

    Gazarian, Karlen G; Solis, Carlos F; Gazarian, Tatiana G; Rowley, Merrill; Laclette, Juan P

    2012-03-01

    Paramyosin of the pig-human parasite Taenia solium (TPmy) is a α-helical protein located on the worm surface that is suggested to fulfill an immunomodulatory role protecting the parasite against host immune system. Besides, in challenging experiments the protein shows a vaccine potential. These observations imply that TPmy harbors antigenic determinants for each of these contrasting actions. However the suggestion was not given a support from experimental data because respective epitopes have not been described thus far. To circumvent this difficulty, we use synthetic peptides with sequences of regions composed of α-helical or linear structure to induce rabbit antibody responses for phage-display mapping of epitope core amino-acid sets. Antibodies to α-helical regions were weak binders and M13 phage-displayed peptides selected by them from two different libraries exhibited no amino-acid similarities with the original protein site. In contrast, the antibodies produced in response to non-helical segment within α-helical structure were better binders and selectors of perfect structural mimics of the protein site. This first phage display epitope analysis of TPmy supports the notion that the rod-like α-helix, which encompasses over 90% of the total amino acids, may serve as an immunomodulatory shield that protects the parasite. Further, the seven non-helical segments of the TPmy molecule may represent the only anti-parasite discrete immunogenic epitopes whose representative mimotopes can be utilized in development of pure epitope vaccines. PMID:22015270

  11. Vaccine potential of meningococcal FrpB: studies on surface exposure and functional attributes of common epitopes.

    PubMed

    Ala'Aldeen, D A; Davies, H A; Borriello, S P

    1994-05-01

    Neisseria meningitidis expresses several novel outer membrane proteins (OMPs) in vivo and when grown under iron limitation in vitro. One of the most prominent is a 70 kDa iron-regulated protein (FrpB). FrpB was purified by elution from SDS-polyacrylamide gels and rabbit polyclonal antiserum (R-70) was raised against it. R-70 was bactericidal against homologous, but not heterologous, strains in the presence of human complement. The bactericidal activity was retained when R-70 was adsorbed with formaldehyde-fixed iron-replete cells (i.e. not expressing FrpB), but lost when absorbed with fixed iron-restricted cells (which express FrpB). A murine monoclonal anti-FrpB antibody (mAb M70) was raised against a common epitope which showed complete cross-reaction on Western blots of OMPs from other serogroups and serotypes of N. meningitidis and some commensal Neisseriae species. However, it failed to kill the organism. Immunogold electron microscopy on ultrathin sections, using the R-70 antiserum adsorbed with fixed iron-replete cells, showed labelling on 40% of the cells, whereas the R-70 adsorbed with fixed iron-restricted cells and mAb M70 failed to label. However, none of these sera labelled whole cells, suggesting lack of surface accessibility. It appears that the highly conserved cross-reactive epitopes of FrpB only become exposed in the process of generating the antigen, whereas the surface-exposed epitopes recognized in killing assays are immunologically variable among different strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7518627

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

    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

  14. Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

    PubMed

    Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

    2014-01-01

    Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization. PMID:25375337

  15. Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.

    PubMed Central

    Chow, L P; Liu, S L; Yu, C J; Liao, H K; Tsai, J J; Tang, T K

    2000-01-01

    The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein. PMID:10677362

  16. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    PubMed

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  17. Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide

    PubMed Central

    Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A.; Ehrlich, Hartmut J.; Schwarz, Hans P.; Walsh, Dominic M.; Solomon, Alan; O’Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer’s disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ’s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody’s nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody’s lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  18. Conservation physiology

    PubMed Central

    Kronfeld-Schor, Noga

    2014-01-01

    Global change presents a huge and exciting challenge to the study of thermal physiology. The implication of thermoregulatory strategies and abilities for the survival of individuals and species, are of high importance for predicting species response to global change challenges and ways to mitigate them, and for conservation acts. A good example of such a study is the paper by Cooper and Withers in this issue.1

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

    PubMed Central

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

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion. PMID:26261576

  20. Heron conservation

    USGS Publications Warehouse

    2000-01-01

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

  1. The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography.

    PubMed

    Stalder, Elizabeth S; Nagy, Lauren H; Batalla, Pilar; Arthur, Terrance M; Thompson, Nancy E; Burgess, Richard R

    2011-05-01

    Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from five species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the β-subunit of bacterial RNA polymerase. This sequence is located in the "beta-flap" domain of RNA polymerase (and essentially comprises the "flap-tip helix"), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest. PMID:21215316

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

    PubMed Central

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

    2016-01-01

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

  3. Identification of dominant epitopes of synthetic immunocontraceptive vaccines that induce antibodies in dogs.

    PubMed

    Yu, Meng; Zeng, Weiguang; Pagnon, Joanne; Walker, John; Ghosh, Souravi; Wang, Lin-Fa; Jackson, David C

    2005-08-31

    The specificities of immunoglobulin G antibodies obtained from the sera of dogs inoculated with totally synthetic immunocontraceptive vaccine candidates based on luteinising hormone releasing hormone (LHRH: amino acid sequence HWSYGLRPG) were examined using peptides expressed in a phage display library. The three vaccine candidates each contained a different T helper-cell epitope chemically linked with the same LHRH amino acid sequence HWSYGLRPG and all of them elicited high antibody titres against the hormone. Delineation of epitopes recognised by sera from vaccinated dogs using a phage display library indicated that two of the three vaccine candidates induced antibody directed to the consensus sequence xHWSxxLxxx whereas the third vaccine candidate induced antibody against the consensus sequence xxxxxxxRPx. Two of the three vaccine candidates elicited antibodies against B cell epitopes present within the helper T-cell epitope component of the vaccine whereas the third vaccine did not. The occurrence of anti-T helper cell epitope antibodies appeared to have little or no effect on the generation of the anti-LHRH responses indicating that carrier-induced epitope suppression was not operating here. Our results also demonstrated that with animal sera of high quality, it is possible to delineate immunodominant epitopes recognised by polyclonal antibodies with high efficiency using phage display library. The approach has utility in the definition of immunodominant epitopes, which may "decoy" antibody responses away from other epitopes, which may be more useful in prophylaxis or therapy. PMID:15927323

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

    PubMed

    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

  5. Bioinformatics Resources and Tools for Conformational B-Cell Epitope Prediction

    PubMed Central

    Sun, Pingping; Ju, Haixu; Liu, Zhenbang; Ning, Qiao; Zhang, Jian; Zhao, Xiaowei; Huang, Yanxin; Ma, Zhiqiang; Li, Yuxin

    2013-01-01

    Identification of epitopes which invoke strong humoral responses is an essential issue in the field of immunology. Localizing epitopes by experimental methods is expensive in terms of time, cost, and effort; therefore, computational methods feature for its low cost and high speed was employed to predict B-cell epitopes. In this paper, we review the recent advance of bioinformatics resources and tools in conformational B-cell epitope prediction, including databases, algorithms, web servers, and their applications in solving problems in related areas. To stimulate the development of better tools, some promising directions are also extensively discussed. PMID:23970944

  6. The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120

    PubMed Central

    Billings, Erik; Rao, Mangala; Williams, Constance; Zolla-Pazner, Susan; Bailer, Robert T.; Koup, Richard A.; Madnote, Sirinan; Arworn, Duangnapa; Shen, Xiaoying; Tomaras, Georgia D.; Currier, Jeffrey R.; Jiang, Mike; Magaret, Craig; Andrews, Ch