Science.gov

Sample records for epitopes preferentially recognized

  1. Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

    SciTech Connect

    Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

    1986-12-01

    A radioimmunoassay that makes use of whole Schistosomula and /sup 125/I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000.

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

    PubMed

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

    1996-10-15

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

  3. Characterization of Two Novel mAbs Recognizing Different Epitopes on CD43

    PubMed Central

    Kim, Soseul; Hong, Jeong won; Cho, Woon-Dong; Moon, Yoo Ri; Yoon, Sang Soon; Kim, Min-Young; Hong, Kwon Pyo; Lee, Yong-Moon; Yi, Jae Hyuk; Ham, Young Jun; Rah, Hyung Chul; Kim, Seung Ryul

    2014-01-01

    JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43. PMID:24999313

  4. 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

  5. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion.

    PubMed

    Delong, Thomas; Wiles, Timothy A; Baker, Rocky L; Bradley, Brenda; Barbour, Gene; Reisdorph, Richard; Armstrong, Michael; Powell, Roger L; Reisdorph, Nichole; Kumar, Nitesh; Elso, Colleen M; DeNicola, Megan; Bottino, Rita; Powers, Alvin C; Harlan, David M; Kent, Sally C; Mannering, Stuart I; Haskins, Kathryn

    2016-02-12

    T cell-mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D. PMID:26912858

  6. Preferential recognition of epitopes on AGE-IgG by the autoantibodies in rheumatoid arthritis patients.

    PubMed

    Ahmad, Saman; Habib, Safia; Moinuddin; Ali, Asif

    2013-01-01

    Incubation of proteins with glucose lead to their non-enzymatic glycation ultimately resulting in the formation of advanced glycation end products (AGEs) in vivo. AGEs alter unique three dimensional structures of various plasma proteins such as IgG. The role of oxidative stress in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, is well established. In view of this, commercially available human IgG was glycated in vitro with physiological concentration of glucose (5mM) and the possible involvement of glycated IgG (AGE-IgG) in RA was evaluated. The RA patients were divided into two groups on the basis of disease onset with respect to age: group I (early onset: 20-32 years) and group II (late onset: 36-54 years). AGE-IgG and oxidative stress levels were detected in RA patients and normal healthy individuals by nitroblue tetrazolium (NBT) assay and carbonyl content estimation respectively. Binding characteristics and specificity of RA antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). We observed preferential binding of RA antibodies to AGE-IgG in comparison to native IgG. Band shift assay further substantiated the enhanced recognition of AGE-IgG by RA antibodies. The results suggest that glycation of IgG results in the generation of neo-epitopes, making it a potential immunogen. Our findings project AGE-IgG as one of the factors for induction of circulating RA autoantibodies. PMID:23073292

  7. HIV-1 epitope-specific CD8+ T cell responses strongly associated with delayed disease progression cross-recognize epitope variants efficiently.

    PubMed

    Turnbull, Emma L; Lopes, A Ross; Jones, Nicola A; Cornforth, David; Newton, Phillipa; Aldam, Diana; Pellegrino, Pierre; Turner, Jo; Williams, Ian; Wilson, Craig M; Goepfert, Paul A; Maini, Mala K; Borrow, Persephone

    2006-05-15

    The ability of HIV-1-specific CD8(+) T cell responses to recognize epitope variants resulting from viral sequence variation in vivo may affect the ease with which HIV-1 can escape T cell control and impact on the rate of disease progression in HIV-1-infected humans. Here, we studied the functional cross-reactivity of CD8 responses to HIV-1 epitopes restricted by HLA class I alleles associated with differential prognosis of infection. We show that the epitope-specific responses exhibiting the most efficient cross-recognition of amino acid-substituted variants were those strongly associated with delayed progression to disease. Not all epitopes restricted by the same HLA class I allele showed similar variant cross-recognition efficiency, consistent with the hypothesis that the reported associations between particular HLA class I alleles and rate of disease progression may be due to the quality of responses to certain "critical" epitopes. Irrespective of their efficiency of functional cross-recognition, CD8(+) T cells of all HIV-1 epitope specificities examined showed focused TCR usage. Furthermore, interpatient variability in variant cross-reactivity correlated well with use of different dominant TCR Vbeta families, suggesting that flexibility is not conferred by the overall clonal breadth of the response but instead by properties of the dominant TCR(s) used for epitope recognition. A better understanding of the features of T cell responses associated with long-term control of viral replication should facilitate rational vaccine design. PMID:16670322

  8. 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

  9. 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

  10. 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

  11. Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

    PubMed Central

    Delong, Thomas; Wiles, Timothy A.; Baker, Rocky L.; Bradley, Brenda; Barbour, Gene; Reisdorph, Richard; Kumar, Nitesh; Elso, Colleen M.; Armstrong, Michael; Powell, Roger L.; Reisdorph, Nichole; DeNicola, Megan; Bottino, Rita; Powers, Alvin C.; Harlan, David M.; Kent, Sally C.; Mannering, Stuart I.; Haskins, Kathryn

    2016-01-01

    Type 1 diabetes (T1D) is caused by T cell mediated destruction of the insulin-producing β cells. CD4 T cell responses play a central role in β-cell destruction but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. To address this we used a panel of diabetes triggering CD4 T cell clones isolated from non-obese diabetic (NOD) mice. Here we show that these pathogenic CD4 T cells target peptide ligands that are formed by covalent crosslinking of proinsulin peptides to other peptides present in β-cell secretory granules. These hybrid insulin peptides (HIPs) are highly antigenic for CD4 T cells and can be detected by mass spectrometry in β-cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. The discovery that autoreactive T cells target hybrid peptides may explain how immune tolerance is broken in T1D. PMID:26912858

  12. Melanoma-specific CD4+ T cells recognize nonmutated HLA-DR-restricted tyrosinase epitopes.

    PubMed

    Topalian, S L; Gonzales, M I; Parkhurst, M; Li, Y F; Southwood, S; Sette, A; Rosenberg, S A; Robbins, P F

    1996-05-01

    Tyrosinase was the first melanoma-associated antigen shown to be recognized by CD4+ T cells. In this study, we have identified two HLA-DRB1*0401-restricted peptides recognized by these T cells: Ty 56-70 and Ty 448-462. As with many of the MHC class I-restricted melanoma epitopes, both are nonmutated self peptides that have intermediate and weak MHC binding affinities, respectively. Mutated and truncated versions of these peptides were used to define their MHC binding anchor residues. Anchor residues were then modified to derive peptides with increased MHC binding affinities and T cell stimulatory properties. Ty 56-70 and Ty 448-462 enhance the list of immunogenic HLA-A2-, A24-, and B44-restricted tyrosinase peptides already described. Thus, tyrosinase provides a model for anti-melanoma vaccines in which a single molecule can generate multivalent immunization incorporating both CD4+ and CD8+ T cell responses. PMID:8642306

  13. A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

    PubMed Central

    Fischer, D C; Haubeck, H D; Eich, K; Kolbe-Busch, S; Stöcker, G; Stuhlsatz, H W; Greiling, H

    1996-01-01

    Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan. PMID:8836155

  14. A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

    PubMed

    Fischer, D C; Haubeck, H D; Eich, K; Kolbe-Busch, S; Stöcker, G; Stuhlsatz, H W; Greiling, H

    1996-09-15

    Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan. PMID:8836155

  15. 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

  16. 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

  17. Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

    PubMed Central

    Barthe, C; Joly, J R; Ramsay, D; Boissinot, M; Benhamou, N

    1988-01-01

    Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing. Images PMID:2454935

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. 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

  1. 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

  2. Critical Epitopes in the Nucleocapsid Protein of SFTS Virus Recognized by a Panel of SFTS Patients Derived Human Monoclonal Antibodies

    PubMed Central

    Yu, Li; Zhang, Li; Sun, Lina; Lu, Jing; Wu, Wei; Li, Chuan; Zhang, Quanfu; Zhang, Fushun; Jin, Cong; Wang, Xianjun; Bi, Zhenqiang; Li, Dexin; Liang, Mifang

    2012-01-01

    Background SFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. Methodology/Principal Findings We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. Conclusions/Significance The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection. PMID:22719874

  3. 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

  4. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

    PubMed Central

    Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

    2012-01-01

    Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

  5. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

    PubMed

    Gilman, Morgan S A; Moin, Syed M; Mas, Vicente; Chen, Man; Patel, Nita K; Kramer, Kari; Zhu, Qing; Kabeche, Stephanie C; Kumar, Azad; Palomo, Concepción; Beaumont, Tim; Baxa, Ulrich; Ulbrandt, Nancy D; Melero, José A; Graham, Barney S; McLellan, Jason S

    2015-07-01

    Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. PMID:26161532

  6. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

    PubMed Central

    Gilman, Morgan S. A.; Moin, Syed M.; Mas, Vicente; Chen, Man; Patel, Nita K.; Kramer, Kari; Zhu, Qing; Kabeche, Stephanie C.; Kumar, Azad; Palomo, Concepción; Beaumont, Tim; Baxa, Ulrich; Ulbrandt, Nancy D.; Melero, José A.; Graham, Barney S.; McLellan, Jason S.

    2015-01-01

    Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. PMID:26161532

  7. Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

    PubMed Central

    Austin, S. Kyle; Dowd, Kimberly A.; Shrestha, Bimmi; Nelson, Christopher A.; Edeling, Melissa A.; Johnson, Syd; Pierson, Theodore C.; Diamond, Michael S.; Fremont, Daved H.

    2012-01-01

    We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development. PMID:23055922

  8. 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

  9. Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies1[S

    PubMed Central

    Tsiantoulas, Dimitrios; Perkmann, Thomas; Afonyushkin, Taras; Mangold, Andreas; Prohaska, Thomas A.; Papac-Milicevic, Nikolina; Millischer, Vincent; Bartel, Caroline; Hörkkö, Sohvi; Boulanger, Chantal M.; Tsimikas, Sotirios; Fischer, Michael B.; Witztum, Joseph L.; Lang, Irene M.; Binder, Christoph J.

    2015-01-01

    Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA+ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE+ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD. PMID:25525116

  10. Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells

    PubMed Central

    Seiler, Till; Woelfle, Manuela; Yancopoulos, Sophia; Catera, Rosa; Li, Wentian; Hatzi, Katerina; Moreno, Carol; Torres, Marcela; Paul, Santanu; Dohner, Hartmut; Stilgenbauer, Stephan; Kaufman, Matthew S.; Kolitz, Jonathan E.; Allen, Steven L.; Rai, Kanti R.; Chu, Charles C.

    2009-01-01

    Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL–derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL. PMID:19690339

  11. CMV-Specific T-cells Generated From Naïve T-cells Recognize Atypical Epitopes And May Be Protective in Vivo

    PubMed Central

    Hanley, Patrick J.; Melenhorst, Jan J.; Nikiforow, Sarah; Scheinberg, Phillip; Blaney, James W.; Demmler-Harrison, Gail; Cruz, C. Russell; Lam, Sharon; Krance, Robert A.; Leung, Kathryn S.; Martinez, Caridad A.; Liu, Hao; Heslop, Helen E.; Rooney, Cliona M.; Shpall, Elizabeth J.; Barrett, A. John; Rodgers, John R.; Bollard, Catherine M.

    2015-01-01

    Adoptive transfer of adult-seropositive, cytomegalovirus (CMV)-specific T-cells can effectively restore antiviral immunity after transplantation. Lack of CMV-specific memory T-cells in blood from CMV-seronegative adult and cord blood (CB) donors restricts the availability of donor-derived virus-specific T-cells for immunoprophylaxis. Here we demonstrate the feasibility of naïve-donor-derived CMV-specific T-cell therapy for transplant recipients. Primed naïve T-cells recognized only atypical epitopes and with a similar avidity to CMV-seropositive-derived T-cells recognizing typical epitopes, but T-cells from CMV-seropositive donors recognizing atypical epitopes had a lower avidity suggesting the loss of high-avidity T-cells over time. Clonotypic analysis revealed T-cells recognizing atypical CMVpp65 epitopes in the peripheral blood of recipients of CB grafts who did not develop CMV. T-cell receptors from atypical epitopes were most common in unmanipulated CB units explaining why these T-cells expanded. When infused to recipients, naïve donor-derived virus specific T-cells that recognized atypical epitopes were associated with prolonged periods of CMV-free survival and complete remission. PMID:25925682

  12. 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

  13. 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

  14. A unique phenotype of skin-associated lymphocytes in humans. Preferential expression of the HECA-452 epitope by benign and malignant T cells at cutaneous sites.

    PubMed Central

    Picker, L. J.; Michie, S. A.; Rott, L. S.; Butcher, E. C.

    1990-01-01

    It has been proposed that the skin is a functionally unique compartment of the immune system, although little direct evidence supporting this hypothesis has been presented. Here we show that lymphocyte populations at cutaneous sites can be differentiated from otherwise similar populations at noncutaneous sites by their preferential expression of an epitope defined by the MAb HECA-452. This MAb recognizes a predominantly 200-kd cell-surface glycoprotein present on about 16% of peripheral blood T cells, including both CD4+ and CD8+ T cells (17% and 11% HECA-452+, respectively), as well as TCR-delta-bearing T cells (32%+). Most thymocytes (99%) lacked HECA-452 antigen expression, and essentially all the HECA-452+ peripheral blood T cells were found in the adhesion molecule high, CD45R low putative memory cell subset, findings suggesting that HECA-452 expression develops peripherally as a consequence of antigenic stimulation. However, the HECA-452 antigen is not a conventional activation antigen because it was not upregulated with mitogen stimulation of peripheral blood T cells. Most significantly, among 54 diverse specimens of normal/reactive lymphoid tissues and sites of chronic inflammation, there was a clear association of lymphocyte HECA-452 expression and cutaneous location. In extracutaneous sites (n = 38) only about 5% of lymphocytes within the T-cell areas of these tissues expressed this antigen, whereas in inflammatory skin lesions (n = 16), 85% were HECA-452+. The association of HECA-452 expression and cutaneous location was also seen in a series of T-cell lymphomas. The malignant cells of 16 of 18 cases of epidermotropic (patch/plaque) stage mycosis fungoides were HECA-452+, as well as 2 of 7 nonmycosis fungoides peripheral T-cell lymphomas in skin. In contrast, this antigen was not expressed in thymic (lymphoblastic) lymphomas (n = 14), nonepidermotropic (tumor) stage mycosis fungoides (n = 5), and noncutaneous peripheral T-cell lymphomas (n = 15). Among

  15. Astrocytes and microglia in human brain share an epitope recognized by a B-lymphocyte-specific monoclonal antibody (LN-1).

    PubMed Central

    Dickson, D. W.; Mattiace, L. A.

    1989-01-01

    A B-lymphocyte-specific mouse monoclonal antibody, LN-1, recognizes two morphologic classes of glial cells in human brain. The nature and duration of tissue fixation and processing are critical in the detection of the two cell types. In tissue that is lightly fixed, LN-1 recognizes astrocytes. The astrocytic nature of the LN-1 reactive glial cell was confirmed by cytologic features, tissue distribution, immunoelectron microscopy, double labeling immunofluorescent microscopy, and staining of serial sections with antibodies to glial fibrillary acidic protein. In tissue that is fixed for longer periods or in Bouin's fixative, two glial cell types are recognized: astrocytes and microglia. The identity of the latter cell type as microglia was confirmed by morphologic features, tissue distribution, immunoelectron microscopy, and double staining with monoclonal antibodies or lectins to macrophage markers, including class II major histocompatibility antigens. The two cell types had different disposition in senile plaques of elderly individuals and of those with Alzheimer's disease. Astrocytes were present at the periphery of the plaques, whereas microglial cells were centrally placed, often in juxtaposition to amyloid. The results are discussed with respect to ontogeny of glial cells and the ability of monoclonal antibodies to recognize epitopes on unrelated proteins. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:2476034

  16. Structural Characterization of Viral Epitopes Recognized by Broadly Cross-Reactive Antibodies

    PubMed Central

    Lee, Peter S.; Wilson, Ian A.

    2015-01-01

    Influenza hemagglutinin (HA) is the major surface glycoprotein on influenza viruses and mediates viral attachment and subsequent fusion with host cells. The HA is the major target of the immune response, but due to its high level of variability, as evidenced by substantial antigenic diversity, it had been historically considered to elicit only a narrow, strain-specific antibody response. However, a recent explosion in the discovery of broadly neutralizing antibodies (bnAbs) to influenza virus has identified two major supersites of vulnerability on the HA through structural characterization of HA-antibody complexes. These commonly targeted epitopes are involved with receptor binding as well as the fusion machinery and, hence, are functionally conserved and less prone to mutation. These bnAbs can neutralize viruses by blocking infection or the spread of infection by preventing progeny release. Structural analyses of these bnAbs show they exhibit striking similarities and trends in recognition of the HA and use recurring recognition motifs, despite substantial differences in their germline genes. This information can be utilized in design of novel therapeutics as well as in immunogens for improved vaccines with greater breadth and efficacy. PMID:25037260

  17. Epitope mapping of monoclonal antibody 4C8 recognizing the protein huntingtin.

    PubMed

    Cong, Shu-Yan; Pepers, Barry A; Roos, Raymund A C; Van Ommen, Gert-Jan B; Dorsman, Josephine C

    2005-10-01

    Huntington's disease is a dominantly inherited, devastating neurodegenerative disorder, caused by a polyglutamine expansion (>37) in the N-terminal region of huntingtin, a protein of unknown function. In patients and normal individuals, N-terminal fragments of huntingtin are found, and the N-terminal fragments of mutant huntingtin are cytotoxic. The functions of wild-type huntingtin and the mechanisms underlying the toxic effects of mutant huntingtin are still ill defined. To get more insight into these topics, monoclonal antibodies (MAbs) are indispensable tools. Antibodies raised against the N-terminus are especially important. Among these, the 4C8 mouse MAb has been extensively used in various approaches. In this study, we have mapped the epitope of 4C8 to a 15-amino acid (aa) region spanning from aa 443 to 457 of the human protein, and found that mutation of three consecutive glutamic acids present in this region disrupts the recognition by 4C8. These results allow a more accurate interpretation of the results obtained by usage of the 4C8 antibody and broaden the utility of this antibody. PMID:16225422

  18. 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

  19. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5

    PubMed Central

    Xia, Lin; Xian, Yangfei; Wang, Daning; Chen, Yuanzhi; Huang, Xiaofen; Bi, Xingjian; Yu, Hai; Fu, Zheng; Liu, Xinlin; Li, Shaowei; An, Zhiqiang; Luo, Wenxin; Zhao, Qinjian; Xia, Ningshao

    2016-01-01

    The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr135 and Val141 on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection. PMID:26750243

  20. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5.

    PubMed

    Xia, Lin; Xian, Yangfei; Wang, Daning; Chen, Yuanzhi; Huang, Xiaofen; Bi, Xingjian; Yu, Hai; Fu, Zheng; Liu, Xinlin; Li, Shaowei; An, Zhiqiang; Luo, Wenxin; Zhao, Qinjian; Xia, Ningshao

    2016-01-01

    The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr(135) and Val(141) on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection. PMID:26750243

  1. Characterization of a monoclonal antibody recognizing an epitope designated as canine leukocyte-associated antigen.

    PubMed

    Yang, W C; Esquenazi, V; Carreno, M; Vallone, T; Fuller, L; Roth, D; Nery, J; Burke, G; Miller, J

    1994-07-27

    An IgG1 monoclonal antibody (mAb), designated as 15F1.5, was generated against surface determinants of a dog peripheral blood-derived PHA-induced IL-2-dependent T cell line. It reacted with 65-80% of peripheral blood mononuclear cells (PBMCs), 90-95% of polymorphonuclear cells (PMNs), 65-70% of thymocytes, 85-95% of Thy-1 positive cells and 85-95% of IL-2-dependent T lymphoid cells in flow cytometry. It was nonreactive with peripheral blood red cells and platelets. It immunoprecipitated 95 and 150 Kd proteins derived from detergent solubilized lymphocyte membranes. Indirect immunofluorescent and immunoperoxidase staining of frozen tissue sections demonstrated positive reactivity to cells in lymphoid but not nonlymphoid tissues. The 15F1.5 antibody was not directly mitogenic for PBMC's. It caused significant decrease (P < or = 0.05) in the lymphoproliferative response to T-dependent B cell mitogens, such as pokeweed mitogen (PWM) and staphage lysate (SPL), without significant effects on responses to the T cell mitogens, phytohemagglutinin (PHA), and concanavalin A (Con A). The mixed lymphocyte culture (MLC) response and both the proliferative and effector arms of the cell-mediated cytotoxicity reactions (CMC) were inhibited in a dose-dependent manner. The mAb also inhibited the auto- and allolymphoproliferative reactivity of mixed lymphocyte kidney or islet cell cultures (MLKC and MLIC), and the adhesion of T lymphoblasts and PMA-treated PMNs to endothelial cells. In vivo administration of the 15F1.5 (20 mg/day for 5 days) caused an immediate and prolonged reduction in MLC responses, associated with cell binding of the mAb to PBMC and epitope modulation during the course of treatment, as indicated by flow cytometry. These results suggest that 15F1.5 is an immunomodulating antibody reacting with canine LFA-1. Thus, this mAb would be useful in studying the role of LFA-1/ICAM-1 in graft rejection as well as other inflammatory responses. It would also allow the use of

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. 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

  5. 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

  6. The novel cuticular collagen Ovcol-1 of Onchocerca volvulus is preferentially recognized by immunoglobulin G3 from putatively immune individuals.

    PubMed Central

    Stewart, G R; Zhu, Y; Parredes, W; Tree, T I; Guderian, R; Bradley, J E

    1997-01-01

    The cDNA sequence encoding an Onchocerca volvulus collagen, Ovcol-1, has been isolated and the corresponding native antigen has been identified. The cDNA encodes an open reading frame of 96 amino acid residues containing an uninterrupted 66-residue Gly-X-Y repeat triple-helical (TH) domain (where X and Y may be any amino acids) flanked by a 26-residue amino non-TH domain and a 4-residue carboxyl non-TH domain. The size (9.7 kDa) and structure of the deduced molecule are unique among previously identified collagen chains. This novel collagen type has been designated "mini-chain collagen." Native Ovcol-1 is aqueous soluble and resolves by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 14.2 kDa under reducing conditions. Immunoelectron microscopy of adult female O. volvulus localized Ovcol-1 to the cuticles of both the adult worm and uterine microfilaria. A group of individuals from an area in Ecuador where O. volvulus is hyperendemic have been classified as putatively immune (PI) to O. volvulus infection. Analysis of the humoral immune responses to Ovcol-1 demonstrated that immunoglobulin G3 (IgG3) of PI individuals preferentially recognized this antigen in comparison to IgG3 of infected individuals. PMID:8975907

  7. 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

  8. 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

  9. 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

  10. Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope.

    PubMed

    Ikeda, Masahiro; Yamaguchi, Miki; Kato, Kazunori; Nakamura, Kiminori; Shiina, Sagano; Ichikawa-Ando, Takako; Misaka, Hirofumi; Myojo, Kensuke; Nakamura, Kazuyasu; Sugimoto, Yoshiyuki; Hamada, Hirofumi

    2015-03-20

    TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments. PMID:25701778

  11. Proteomic Characterization of Helicobacter pylori CagA Antigen Recognized by Child Serum Antibodies and Its Epitope Mapping by Peptide Array

    PubMed Central

    Akada, Junko; Okuda, Masumi; Hiramoto, Narumi; Kitagawa, Takao; Zhang, Xiulian; Kamei, Shuichi; Ito, Akane; Nakamura, Mikiko; Uchida, Tomohisa; Hiwatani, Tomoko; Fukuda, Yoshihiro; Nakazawa, Teruko; Kuramitsu, Yasuhiro; Nakamura, Kazuyuki

    2014-01-01

    Serum antibodies against pathogenic bacteria play immunologically protective roles, and can be utilized as diagnostic markers of infection. This study focused on Japanese child serum antibodies against Helicobacter pylori, a chronically-infected gastric bacterium which causes gastric cancer in adults. Serological diagnosis for H. pylori infection is well established for adults, but it needs to be improved for children. Serum samples from 24 children, 22 H. pylori (Hp)-positive and 2 Hp-negative children, were used to catalogue antigenic proteins of a Japanese strain CPY2052 by two-dimensional electrophoresis followed by immunoblot and LC-MS/MS analysis. In total, 24 proteins were identified as candidate antigen proteins. Among these, the major virulence factor, cytotoxin-associated gene A protein (CagA) was the most reactive antigen recognized by all the Hp-positive sera even from children under the age of 3 years. The major antigenic part of CagA was identified in the middle region, and two peptides containing CagA epitopes were identified using a newly developed peptide/protein-combined array chip method, modified from our previous protein chip method. Each of the epitopes was found to contain amino acid residue(s) unique to East Asian CagA. Epitope analysis of CagA indicated importance of the regional CagA antigens for serodiagnosis of H. pylori infection in children. PMID:25141238

  12. Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection.

    PubMed

    Barzu, S; Nato, F; Rouyre, S; Mazie, J C; Sansonetti, P; Phalipon, A

    1993-09-01

    The invasion plasmid antigen B (IpaB), a 62-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells, is the bacterial antigen most strongly and consistently recognized by the host during infection. The strong systemic and mucosal immune responses observed against this invasin prompted us to map its B-cell epitopes. For this purpose, IpaB was first overexpressed in Shigella flexneri and used to raise rabbit polyclonal antiserum and murine monoclonal antibodies, which were subsequently used to screen a lambda gt11 ipaB library. Inserts of recombinant DNA clones that were specifically recognized by the antisera and antibodies were sequenced, and three distinct determinants were identified. Further characterization of these determinants showed that they were recognized by sera from patients convalescent from shigellosis, suggesting that they are relevant to the humoral response during natural infection. Moreover, the IpaB region comprising the three determinants was systematically recognized by all sera from infected patients that we tested, whereas other regions of the protein were not. These data suggest that this region, located between amino acid residues 147 and 258, is the major immunogenic domain of the invasin in the course of natural infection. PMID:7689541

  13. Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

    PubMed Central

    Moiola, L; Karachunski, P; Protti, M P; Howard, J F; Conti-Tronconi, B M

    1994-01-01

    We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above. Images PMID:7510715

  14. Mapping an epitope in EBNA‐1 that is recognized by monoclonal antibodies to EBNA‐1 that cross‐react with dsDNA

    PubMed Central

    Yadav, Pragya; Carr, Matthew T.; Yu, Ruby; Mumbey‐Wafula, Alice

    2016-01-01

    Abstract Introduction The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen‐I (EBNA‐1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA‐1, 3D4, and demonstrated that it cross‐reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA‐1. The aim of the present study was to characterize another antibody to EBNA‐1 that cross‐reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA‐1 that is recognized by these cross‐reactive antibodies. Methods We generated an IgM MAb to EBNA‐1, 16D2, from EBNA‐1 injected mice and demonstrated by ELISA that it cross‐reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA‐1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain. Results Sequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA‐1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA‐1. Conclusion We have demonstrated that two MAbs that bind EBNA‐1 and cross‐react with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE. PMID:27621818

  15. Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells.

    PubMed

    Hastings, Andrew K; Gilchuk, Pavlo; Joyce, Sebastian; Williams, John V

    2016-05-23

    Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8(+) T cell (TCD8) epitopes, with M39-47 (M39) immunodominant. Tetramer staining detected M39-specific TCD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that TCD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies. PMID:27105560

  16. Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid

    SciTech Connect

    Löfling, Jonas; Michael Lyi, Sangbom; Parrish, Colin R.; Varki, Ajit

    2013-05-25

    Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells. - Highlights: ► Feline and canine parvoviruses recognize Neu5Gc but not Neu5Ac, which differ by one oxygen atom. ► The underlying linkage of these sialic acids does not affect recognition. ► Induced Neu5Gc expression on target cells that normally express Neu5Ac did not enhance infection. ► Thus, the conserved binding preference plays an important yet unknown role in in vivo infections. ► Population and breed variations in Neu5Gc expression occur, likely by regulating the gene CMAH.

  17. A Broadly Flavivirus Cross-Neutralizing Monoclonal Antibody that Recognizes a Novel Epitope within the Fusion Loop of E Protein

    PubMed Central

    Jiang, Tao; Wang, Hua-Jing; Yang, Hai-ou; Tan, Weng-Long; Liu, Ran; Yu, Man; Ge, Bao-Xue; Zhu, Qing-Yu; Qin, E-De; Guo, Ya-Jun; Qin, Cheng-Feng

    2011-01-01

    Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1–4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the 98DRXW101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1–4, YFV, and WNV and confers protection from lethal challenge with DENV 1–4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans. PMID:21264311

  18. The 3' portion of the gene for a Plasmodium yoelii merozoite surface antigen encodes the epitope recognized by a protective monoclonal antibody.

    PubMed Central

    Burns, J M; Daly, T M; Vaidya, A B; Long, C A

    1988-01-01

    The 230-kDa merozoite antigen of the murine malarial parasite Plasmodium yoelii provides a potential model system for the development of a protective erythrocytic stage vaccine. To characterize this antigen at the molecular level, isolated P. yoelii 17XL DNA was used to construct a genomic library in the expression vector lambda gt11. A monoclonal antibody, mAb 302, which passively protected mice against P. yoelii challenge infection, was used to identify a lambda gt11 recombinant clone encoding a portion of the 230-kDa antigen of this parasite. Using this clone as a probe, we identified an mRNA of 7.6 kilobases by RNA blot analysis. Nucleic acid sequence analysis of the clone showed that the epitope recognized by the protective mAb 302 is encoded by the 3' portion of the gene for the 230-kDa antigen. The deduced amino acid sequence revealed that this antigen also contains the tandemly repeated tetrapeptide Gly-Ala-Val-Pro, a series of 10 cysteine residues located within the terminal 110 amino acids, and a potential membrane anchor of 18 hydrophobic residues. Comparison of this C-terminal sequence with the carboxyl segment of the 195-kDa merozoite antigen of Plasmodium falciparum revealed nucleic acid and amino acid sequence similarities ranging from 40% to 70%. The localization of a B-cell epitope recognized by the protective mAb 302 to this carboxyl region of the P. yoelii antigen, combined with the limited strain variability in this region of the homologous 195-kDa antigen of P. falciparum, has implications for the development of an effective erythrocytic stage malarial vaccine. Images PMID:2448778

  19. Canine and feline parvoviruses preferentially recognize the non-human cell surface sialic acid N-glycolylneuraminic acid.

    PubMed

    Löfling, Jonas; Lyi, Sangbom Michael; Parrish, Colin R; Varki, Ajit

    2013-05-25

    Feline panleukopenia virus (FPV) is a pathogen whose canine-adapted form (canine parvovirus (CPV)) emerged in 1978. These viruses infect by binding host transferrin receptor type-1 (TfR), but also hemagglutinate erythrocytes. We show that hemagglutination involves selective recognition of the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) but not N-acetylneuraminic acid (Neu5Ac), which differs by only one oxygen atom from Neu5Gc. Overexpression of α2-6 sialyltransferase did not change binding, indicating that both α2-3 and α2-6 linkages are recognized. However, Neu5Gc expression on target cells did not enhance CPV or FPV infection in vitro. Thus, the conserved Neu5Gc-binding preference of these viruses likely plays a role in the natural history of the virus in vivo. Further studies must clarify relationships between virus infection and host Neu5Gc expression. As a first step, we show that transcripts of CMAH (which generates Neu5Gc from Neu5Ac) are at very low levels in Western dog breed cells. PMID:23497940

  20. The Thai Phase III Trial (RV144) Vaccine Regimen Induces T Cell Responses that Preferentially Target Epitopes within the V2 Region of HIV-1 Envelope

    PubMed Central

    de Souza, Mark S.; Ratto-Kim, Silvia; Chuenarom, Weerawan; Schuetz, Alexandra; Chantakulkij, Somsak; Nuntapinit, Bessara; Valencia-Micolta, Anais; Thelian, Doris; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Paris, Robert M.; Kaewkungwal, Jaranit; Michael, Nelson L.; Rerks-Ngarm, Supachai; Mathieson, Bonnie; Marovich, Mary; Currier, Jeffrey R.; Kim, Jerome H.

    2012-01-01

    The Thai HIV phase III prime-boost trial (RV144) using ALVAC-HIV® (vCP1521) and AIDSVAX B/E® was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT were performed on PBMC from HIV-1 uninfected vaccine (N=61) and placebo (N=10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4+ T cell mediated. Responses were targeted within the HIV Env region, with 15/25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α4β7 integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19/30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4+ T cells, with the majority of responders producing both IL-2 and IFN-γ (12/19; 63%). HIV-Env Ab titers were higher in subjects with IL-2 compared to those without IL-2 secreting HIV-Env specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4+ with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality and functional cytolytic capacity. While the RV144 T cell responses were modest in frequency compared to humoral immune responses, the CD4+ T cell response was directed to HIV-1 Env and more particularly the V2 region. PMID:22529301

  1. Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

    PubMed

    Faleri, Agnese; Santini, Laura; Brier, Sébastien; Pansegrau, Werner; Lo Surdo, Paola; Scarselli, Maria; Buricchi, Francesca; Volpini, Gianfranco; Genovese, Alessia; van der Veen, Stijn; Lea, Susan; Tang, Christoph M; Savino, Silvana; Pizza, Mariagrazia; Finco, Oretta; Norais, Nathalie; Masignani, Vega

    2014-04-01

    Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes. PMID:24371123

  2. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    PubMed

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers. PMID:25996440

  3. New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma

    PubMed Central

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-01-01

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296–390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers. PMID:25996440

  4. 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

  5. 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

  6. 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

  7. Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

    PubMed Central

    Yoshitake, Hiroshi; Hashii, Noritaka; Kawasaki, Nana; Endo, Shuichiro; Takamori, Kenji; Hasegawa, Akiko; Fujiwara, Hiroshi; Araki, Yoshihiko

    2015-01-01

    Ts4, an anti-sperm auto-monoclonal antibody, possesses immunoreactivity to the acrosomal region of mouse epididymal spermatozoa. In addition, the mAb shows specific immunoreactivity to reproduction-related regions such as testicular germ cells and early embryo. Our qualitative study previously showed that the antigen epitope for Ts4 contained a N-linked common oligosaccharide (OS) chain on testicular glycoproteins as determined by Western blotting for testicular glycoproteins after treatment with several glycohydrolases. Since the distribution of the Ts4-epitope is unique, the OS chain in Ts4-epitope may have role(s) in the reproductive process. The aim of this study was to clarify the molecular structure of the Ts4-epitope, particularly its OS moiety. Using Ts4 immunoprecipitation combined with liquid chromatography and multiple-stage mass spectrometry, the candidate carbohydrate structure in the Ts4-epitope is proposed to be N-linked fucosylated agalacto-biantennary with bisecting N-acetylglucosamine (GlcNAc) or with N-acetylgalactosamine-GlcNAc motif. Further binding analyses using various lectins against the mouse testicular Ts4-immunoprecipitants revealed that Phaseolus vulgaris erythroagglutinin and Pisum sativum agglutinin showed positive staining of the bands corresponding to Ts4 reactive proteins. Moreover, the immunoreactivity of Ts4 against the testicular extract was completely abrogated after digestion with β-N-acetylglucosaminidase. These results show that the Ts4-epitope contains agalacto-biantennary N-glycan with bisecting GlcNAc carrying fucose residues. PMID:26222427

  8. Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family

    PubMed Central

    Pollara, Justin; Moody, M. Anthony; Alpert, Michael D.; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B.; Huang, Ying; Gurley, Thaddeus C.; Kozink, Daniel M.; Marshall, Dawn J.; Whitesides, John F.; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H.; Michael, Nelson L.; Tomaras, Georgia D.; Montefiori, David C.; Lewis, George K.; DeVico, Anthony; Evans, David T.; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F.

    2012-01-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs. PMID:22896626

  9. Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

    PubMed

    Bonsignori, Mattia; Pollara, Justin; Moody, M Anthony; Alpert, Michael D; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B; Huang, Ying; Gurley, Thaddeus C; Kozink, Daniel M; Marshall, Dawn J; Whitesides, John F; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H; Michael, Nelson L; Tomaras, Georgia D; Montefiori, David C; Lewis, George K; DeVico, Anthony; Evans, David T; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F

    2012-11-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs. PMID:22896626

  10. 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.

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

    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

  12. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry.

    PubMed

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope. Graphical Abstract ᅟ. PMID:27067900

  13. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  14. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-04-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  15. Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

    PubMed Central

    Jarząb, Anna; Witkowska, Danuta; Ziomek, Edmund; Dąbrowska, Anna; Szewczuk, Zbigniew; Gamian, Andrzej

    2013-01-01

    Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections. PMID:23940590

  16. Shigella flexneri 3a outer membrane protein C epitope is recognized by human umbilical cord sera and associated with protective activity.

    PubMed

    Jarząb, Anna; Witkowska, Danuta; Ziomek, Edmund; Dąbrowska, Anna; Szewczuk, Zbigniew; Gamian, Andrzej

    2013-01-01

    Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections. PMID:23940590

  17. Type 1 Diabetes at-risk children highly recognize Mycobacterium avium subspecies paratuberculosis epitopes homologous to human Znt8 and Proinsulin

    PubMed Central

    Niegowska, Magdalena; Rapini, Novella; Piccinini, Simona; Mameli, Giuseppe; Caggiu, Elisa; Manca Bitti, Maria Luisa; Sechi, Leonardo A.

    2016-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to T1D as a putative environmental agent triggering or accelerating the disease in Sardinian and Italian populations. Our aim was to investigate the role of MAP in T1D development by evaluating levels of antibodies directed against MAP epitopes and their human homologs corresponding to ZnT8 and proinsulin (PI) in 54 T1D at-risk children from mainland Italy and 42 healthy controls (HCs). A higher prevalence was detected for MAP/ZnT8 pairs (62,96% T1D vs. 7,14% HCs; p < 0.0001) compared to MAP/PI epitopes (22,22% T1D vs. 9,52% HCs) and decreasing trends were observed upon time-point analyses for most peptides. Similarly, classical ZnT8 Abs and GADA decreased in a time-dependent manner, whereas IAA titers increased by 12%. Responses in 0–9 year-old children were stronger than in 10–18 age group (75% vs. 69,1%; p < 0.04). Younger age, female sex and concomitant autoimmune disorders contributed to a stronger seroreactivity suggesting a possible implication of MAP in multiple autoimmune syndrome. Cross-reactivity of the homologous epitopes was reflected by a high correlation coefficient (r2 > 0.8) and a pairwise overlap of positivity (>83% for MAP/ZnT8). PMID:26923214

  18. A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies ▿

    PubMed Central

    Pejchal, Robert; Gach, Johannes S.; Brunel, Florence M.; Cardoso, Rosa M.; Stanfield, Robyn L.; Dawson, Philip E.; Burton, Dennis R.; Zwick, Michael B.; Wilson, Ian A.

    2009-01-01

    The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens. PMID:19515770

  19. Characterization of a natural mouse monoclonal antibody recognizing epitopes shared by oxidized low-density lipoprotein and chaperonin 60 of Aggregatibacter actinomycetemcomitans.

    PubMed

    Wang, Chunguang; Kankaanpää, Jari; Kummu, Outi; Turunen, S Pauliina; Akhi, Ramin; Bergmann, Ulrich; Pussinen, Pirkko; Remes, Anne M; Hörkkö, Sohvi

    2016-06-01

    Natural antibodies are predominantly antibodies of the IgM isotype present in the circulation of all vertebrates that have not been previously exposed to exogenous antigens. They are often directed against highly conserved epitopes and bind to ligands of varying chemical composition with low affinity. In this study we cloned and characterized a natural mouse monoclonal IgM antibody selected by binding to malondialdehyde acetaldehyde epitopes on low-density lipoprotein (LDL). Interestingly, the IgM antibody cross-reacted with Aggregatibacter actinomycetemcomitans (Aa) bacteria, a key pathogenic microbe in periodontitis reported to be associated with risk factor for atherosclerosis, thus being named as Aa_Mab. It is more intriguing that the binding molecule of Aa to Aa_Mab IgM was found to be Aa chaperonin 60 or HSP60, a member of heat-shock protein family, behaving not only as a chaperone for correct protein folding but also as a powerful virulence factor of the bacteria for inducing bone resorption and as a putative pathogenic factor in atherosclerosis. The findings will highlight the question of whether molecular mimicry between pathogen components and oxidized LDL could lead to atheroprotective immune activity, and also would be of great importance in potential application of immune response-based preventive and therapeutic strategies against atherosclerosis and periodontal disease. PMID:26786003

  20. Identification of Mono- and Disulfated N-Acetyl-lactosaminyl Oligosaccharide Structures as Epitopes Specifically Recognized by Humanized Monoclonal Antibody HMOCC-1 Raised against Ovarian Cancer*♦

    PubMed Central

    Shibata, Toshiaki K.; Matsumura, Fumiko; Wang, Ping; Yu, ShinYi; Chou, Chi-Chi; Khoo, Kay-Hooi; Kitayama, Kazuko; Akama, Tomoya O.; Sugihara, Kazuhiro; Kanayama, Naohiro; Kojima-Aikawa, Kyoko; Seeberger, Peter H.; Fukuda, Minoru; Suzuki, Atsushi; Aoki, Daisuke; Fukuda, Michiko N.

    2012-01-01

    A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290–298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer. PMID:22194598

  1. CD8(+) T cells remove cysts of Toxoplasma gondii from the brain mostly by recognizing epitopes commonly expressed by or cross-reactive between type II and type III strains of the parasite.

    PubMed

    Ochiai, Eri; Sa, Qila; Perkins, Sara; Grigg, Michael E; Suzuki, Yasuhiro

    2016-01-01

    Our previous study demonstrated that CD8(+) T cells remove cysts of Toxoplasma gondii from the brain through perforin-mediated mechanisms. We here show that a transfer of CD8(+) immune T cells primed with a type II or a type III strain of T. gondii both efficiently removed cysts of a type II strain from infected SCID mice, although the former tended to be slightly more efficient than the latter. Similarly, a transfer of type II-primed CD8(+) T cells removed cysts of a type III strain. Therefore, CD8(+) T cells are capable of removing T. gondii cysts by recognizing epitopes commonly expressed in types II and III strains or cross-reactive between these two genotypes. PMID:27083473

  2. Specificity of antinuclear autoantibodies recognizing the dense fine speckled nuclear pattern: Preferential targeting of DFS70/LEDGFp75 over its interacting partner MeCP2.

    PubMed

    Basu, Anamika; Woods-Burnham, Leanne; Ortiz, Greisha; Rios-Colon, Leslimar; Figueroa, Johnny; Albesa, Roger; Andrade, Luis E; Mahler, Michael; Casiano, Carlos A

    2015-12-01

    Human antinuclear autoantibodies (ANAs) targeting the dense fine speckled (DFS) nuclear protein DFS70, commonly known as lens epithelium derived growth factor p75 (LEDGFp75), present a clinical puzzle since their significance remains elusive. While their frequencies are low in ANA-positive autoimmune rheumatic diseases, they are relatively elevated in clinical laboratory referrals, diverse inflammatory conditions, and 'apparently' healthy individuals. We reported previously that DFS70/LEDGFp75 is an autoantigen in prostate cancer that closely interacts with another 70kD DFS nuclear protein, methyl CpG binding protein 2 (MeCP2). This led us to investigate if anti-DFS sera exclusively target DFS70/LEDGFp75 or also recognize MeCP2. Using several complementary autoantibody detection platforms and cellular/molecular approaches we evaluated 65 human sera producing anti-DFS autoantibodies. Our results show that these antibodies are highly specific for DFS70/LEDGFp75 and do not target MeCP2. Establishing the specificity of anti-DFS autoantibodies has implications for increasing our understanding of their biological significance and clinical utility. PMID:26235378

  3. A galactosyl(alpha 1-3)mannose epitope on phospholipids of Leishmania mexicana and L. braziliensis is recognized by trypanosomatid-infected human sera.

    PubMed Central

    Avila, J L; Rojas, M

    1990-01-01

    An immunoglobulin M antibody reactive with galactosyl(alpha 1-3)mannose [Gal(alpha 1-3)Man] residues present on phospholipids extracted from Leishmania mexicana and L. braziliensis was found to be present in high titer in the serum of every normal individual studied. Periodate oxidation, acid hydrolysis, or acetylation suppressed immunoreactivity, suggesting that an oligosaccharide chain was responsible for antibody binding. Interaction occurs only with alpha-Gal terminal residues, since treatment of purified glycophospholipids with alpha-galactosidase but not with beta-galactosidase abolished it. Antibody bound to galactosyl(alpha 1-3)galactose-linked synthetic antigens but did not bind to the same residues present in rabbit, rat, and guinea pig erythrocytes or in murine laminin. Antigen-antibody binding was strongly blocked with Gal(alpha 1-3)Man and Gal(beta 1-4)Man. These results plus inhibition studies with several oligosaccharides suggest that they are indeed different from antibodies against the galactosyl(alpha 1-3)galactose residue. Anti-Gal(alpha 1-3)Man antibody values were significantly elevated in 89% of patients with diffuse cutaneous leishmaniasis, 84% of patients with localized cutaneous leishmaniasis, 69% of patients with mucocutaneous leishmaniasis, and 44 and 62% of patients with Trypanosoma cruzi or T. rangeli infection, respectively, but not in patients with 15 other different infectious and inflammatory diseases. Anti-Gal(alpha 1-3)Man antibody readily absorbed to American Leishmania and Trypanosoma culture forms, suggesting a surface membrane localization of reactive epitope. Gal(alpha 1-3)Man-bearing glycophospholipid was easily extracted from American Leishmania promastigotes and T. cruzi trypomastigotes as well as from American Trypanosoma culture forms. The possibility that this antibody arises against parasitic glycophospholipid-linked Gal(alpha 1-3)Man terminal residues is proposed. PMID:1696285

  4. 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

  5. The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

    PubMed Central

    Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

    2016-01-01

    The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

  6. The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6.

    PubMed

    Boschert, V; Frisch, C; Back, J W; van Pee, K; Weidauer, S E; Muth, E-M; Schmieder, P; Beerbaum, M; Knappik, A; Timmerman, P; Mueller, T D

    2016-08-01

    The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

  7. Asymptomatic HLA-A*02:01–Restricted Epitopes from Herpes Simplex Virus Glycoprotein B Preferentially Recall Polyfunctional CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect HLA Transgenic Mice against Ocular Herpes

    PubMed Central

    Dervillez, Xavier; Qureshi, Huma; Chentoufi, Aziz A.; Khan, Arif A.; Kritzer, Elizabeth; Yu, David C.; Diaz, Oscar R.; Gottimukkala, Chetan; Kalantari, Mina; Villacres, Maria C.; Scarfone, Vanessa M.; McKinney, Denise M.; Sidney, John; Sette, Alessandro; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2014-01-01

    Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine. PMID:24101547

  8. 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

  9. Brain alpha-dystroglycan displays unique glycoepitopes and preferential binding to laminin-10/11.

    PubMed

    McDearmon, Erin L; Combs, Ariana C; Sekiguchi, Kiyotoshi; Fujiwara, Hironobu; Ervasti, James M

    2006-06-12

    alpha-Dystroglycan was quantitatively enriched from mammalian brain based on its uniform reactivity with Vicia villosa agglutinin and resolved into sub-populations possessing or lacking the sulfated glucuronic acid epitope recognized by monoclonal antibody HNK-1. We generated a new monoclonal antibody specific for a glycoepitope on brain alpha-dystroglycan but absent from alpha-dystroglycan expressed in all other tissues examined. Finally, we found that laminin-10/11 preferentially bound to brain alpha-dystroglycan compared to skeletal muscle alpha-dystroglycan. Our results suggest that tissue-specific glycosylation modifies the laminin binding specificity of alpha-dystroglycan. PMID:16709410

  10. 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

  11. 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

  12. 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

  13. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    PubMed Central

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  14. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

    PubMed

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  15. 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

  16. 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

  17. 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

  18. Recognizing Cataracts

    MedlinePlus

    ... link, please review our exit disclaimer . Subscribe Recognizing Cataracts Watch for Vision Changes as You Age As ... cause of impaired eyesight later in life is cataracts. A cataract is a clouding of the lens ...

  19. 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

  20. 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

  1. Controlled CO preferential oxidation

    DOEpatents

    Meltser, M.A.; Hoch, M.M.

    1997-06-10

    Method is described for controlling the supply of air to a PROX (PReferential OXidation for CO cleanup) reactor for the preferential oxidation in the presence of hydrogen wherein the concentration of the hydrogen entering and exiting the PROX reactor is monitored, the difference there between correlated to the amount of air needed to minimize such difference, and based thereon the air supply to the PROX reactor adjusted to provide such amount and minimize such difference. 2 figs.

  2. HLA-A02:01-restricted epitopes identified from the herpes simplex virus tegument protein VP11/12 preferentially recall polyfunctional effector memory CD8+ T cells from seropositive asymptomatic individuals and protect humanized HLA-A*02:01 transgenic mice against ocular herpes.

    PubMed

    Srivastava, Ruchi; Khan, Arif A; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T; Huang, Jiawei; Scarfone, Vanessa M; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

    2015-03-01

    The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8(+) T cells from HSV-seropositive individuals. However, whether and which VP11/12 epitope-specific CD8(+) T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8(+) T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107(a/b) cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228, and VP11/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RA(low)CCR7(low)CD44(high)CD62L(low)CD27(low)CD28(low)CD8(+) effector memory CD8(+) T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8(+) TEM cell epitopes induced robust and polyfunctional epitope-specific CD8(+) TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8(+) T cells that should guide the development of an effective T cell-based herpes vaccine. PMID:25617474

  3. HLA-A02:01-Restricted Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP11/12 Preferentially Recall Polyfunctional Effector Memory CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect “Humanized” HLA-A*02:01 Transgenic Mice Against Ocular Herpes

    PubMed Central

    Srivastava, Ruchi; Khan, Arif A.; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P.; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T.; Huang, Jiawei; Scarfone, Vanessa M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2014-01-01

    The Herpes Simplex Virus type 1 virion tegument phosphoprotein 11/12 (HSV-1 VP11/12) is a major antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether and which VP11/12-epitope-specific CD8+ T cells play a role in the “natural” protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 716 amino acids sequence of VP11/12. Three out of ten epitopes exhibited high to moderate binding affinity to HLA-A*02:01 molecules. In ten sequentially studied HLA-A*02:01 positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust and polyfunctional effector CD8+ T-cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266–74, VP11/12220–228 and VP11/12702–710. Interestingly, ASYMP individuals had significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory T cells (TEM) specific to the three epitopes, compared to symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of an effective T-cell-based herpes vaccine. PMID:25617474

  4. Induction of Antibodies in Rhesus Macaques That Recognize a Fusion-Intermediate Conformation of HIV-1 gp41

    PubMed Central

    Dennison, S. Moses; Sutherland, Laura L.; Jaeger, Frederick H.; Anasti, Kara M.; Parks, Robert; Stewart, Shelley; Bowman, Cindy; Xia, Shi-Mao; Zhang, Ruijun; Shen, Xiaoying; Scearce, Richard M.; Ofek, Gilad; Yang, Yongping; Kwong, Peter D.; Santra, Sampa; Liao, Hua-Xin; Tomaras, Georgia; Letvin, Norman L.; Chen, Bing; Alam, S. Munir; Haynes, Barton F.

    2011-01-01

    A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope. PMID:22140469

  5. 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

  6. 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

  7. 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

  8. Vaccine Focusing to Cross-Subtype HIV-1 gp120 Variable Loop Epitopes

    PubMed Central

    Cardozo, Timothy; Wang, Shixia; Jiang, Xunqing; Kong, Xiang-Peng; Hioe, Catarina; Krachmarov, Chavdar

    2014-01-01

    We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross-subtype HIV-1 neutralizing Abs can be intentionally elicited in mammals by vaccination. The precise boundaries of the epitopes and conformational flexibility in the presentation of the epitopes in the immunogen appeared to be important for successful elicitation. This work may serve as a starting point for translating the activities of human broadly neutralizing anti-HIV-1 monoclonal antibodies (bNAbs) into matched immunogens that can contribute to an efficacious HIV-1 vaccine. PMID:25045827

  9. 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

  10. Controlled CO preferential oxidation

    DOEpatents

    Meltser, Mark A.; Hoch, Martin M.

    1997-01-01

    Method for controlling the supply of air to a PROX reactor for the preferential oxidation in the presence of hydrogen wherein the concentration of the hydrogen entering and exiting the PROX reactor is monitored, the difference therebetween correlated to the amount of air needed to minimize such difference, and based thereon the air supply to the PROX reactor adjusted to provide such amount and minimize such difference.

  11. 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

  12. 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

  13. 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

  14. 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

  15. Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

    SciTech Connect

    Zolla-Pazner, Susan Cohen, Sandra Sharpe; Krachmarov, Chavdar; Wang, Shixia; Pinter, Abraham; Lu, Shan

    2008-03-15

    Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.

  16. 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

  17. 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

  18. 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

  19. 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

  20. Preferentially Expressed Antigen in Melanoma (PRAME) and the PRAME Family of Leucine-Rich Repeat Proteins.

    PubMed

    Hermes, Nora; Kewitz, Stefanie; Staege, Martin S

    2016-01-01

    Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies. PMID:26694250

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. [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

  9. 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

  10. Preferential Remedies for Employment Discrimination

    ERIC Educational Resources Information Center

    Edwards, Harry T.; Zaretsky, Barry L.

    1975-01-01

    An overview of the problem of preferential remedies to achieve equal employment opportunities for women and minority groups. Contends that "color blindness" will not end discrimination but that some form of "color conscious" affirmative action program must be employed. Temporary preferential treatment is justified, according to the author, by the…

  11. 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

  12. Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.

    PubMed

    Schickli, Jeanne H; Whitacre, David C; Tang, Roderick S; Kaur, Jasmine; Lawlor, Heather; Peters, Cory J; Jones, Joyce E; Peterson, Darrell L; McCarthy, Michael P; Van Nest, Gary; Milich, David R

    2015-04-01

    Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes. PMID:25751145

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

    PubMed

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

    2015-04-15

    Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GAD-specific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes. PMID:25867471

  14. 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

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

    PubMed

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

    2014-11-01

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

  16. Neo-epitopes on methylglyoxal modified human serum albumin lead to aggressive autoimmune response in diabetes.

    PubMed

    Jyoti; Mir, Abdul Rouf; Habib, Safia; Siddiqui, Sheelu Shafiq; Ali, Asif; Moinuddin

    2016-05-01

    Glyco-oxidation of proteins has implications in the progression of diabetes type 2. Human serum albumin is prone to glyco-oxidative attack by sugars and methylglyoxal being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. This study has probed the methylglyoxal mediated modifications of HSA, the alterations in its immunological characteristics and possible role in autoantibody induction. We observed an exposure of chromophoric groups, loss in the fluorescence intensity, generation of AGEs, formation of cross-linked products, decrease in α-helical content, increase in hydrophobic clusters, FTIR band shift, attachment of methylglyoxal to HSA and the formation of N(ε)-(carboxyethyl) lysine in the modified HSA, when compared to the native albumin. MG-HSA was found to be highly immunogenic with additional immunogenicity invoking a highly specific immune response than its native counterpart. The binding characteristics of circulating autoantibodies in type 2 diabetes mellitus (DM) patients showed the generation of anti-MG-HSA auto-antibodies in the these patients, that are preferentially recognized by the modified albumin. We propose that MG induced structural perturbations in HSA, result in the generation of neo-epitopes leading to an aggressive auto-immune response and may contribute to the immunopathogenesis of diabetes type 2 associated complications. PMID:26861824

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-04-01

    It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage. PMID:26581676

  19. The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis

    PubMed Central

    KELLY, C G; BOOTH, V; KENDAL, H; SLANEY, J M; CURTIS, M A; LEHNER, T

    1997-01-01

    Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the j3 component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpRl. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784–1130 of PrpRl and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907–931 of PrpRl, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934–1042. The haemagglutinating epitope was mapped to residues 1073–1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpRl may provide a means of preventing infection with P. gingivalis. PMID:9367414

  20. 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

  1. Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    PubMed Central

    Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

    2012-01-01

    Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

  2. Human self protein CD8+ T cell epitopes are both positively and negatively selected

    PubMed Central

    almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-01-01

    The cellular immune system recognizes self epitopes in the context of MHC-I molecules. The immunological general view presumes that these self epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self proteins types. Proteins presenting many epitopes include for example AIRE upregulated Tissue specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less “useful” for the immune system, such as low expression level proteins, are under presented. We combine our epitope estimate with SNP measures to show that this selection can be directly observed through the fraction of non-synonymous SNPs (replacement fraction), which is significantly higher inside epitopes than outside PMID:19291702

  3. Human self-protein CD8+ T-cell epitopes are both positively and negatively selected.

    PubMed

    Almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-04-01

    The cellular immune system recognizes self-epitopes in the context of MHC-I molecules. The immunological general view presumes that these self-epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self-protein types. Proteins presenting many epitopes include, for example, autoimmune regulator (AIRE) upregulated tissue-specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less "useful" for the immune system, such as low expression level proteins, are under-presented. We combine our epitope estimate with single nucleotide polymorphism (SNP) measures to show that this selection can be directly observed through the fraction of non-synonymous SNP (replacement fraction), which is significantly higher inside epitopes than outside. PMID:19291702

  4. Insights into the biological features of the antigenic determinants recognized by four monoclonal antibodies in redia and adult stages of the liver fluke Fasciola hepatica.

    PubMed

    Alba, Annia; Sánchez, Jorge; Hernández, Hilda; Mosqueda, Maryani; Rodríguez, Suanel Y; Capó, Virginia; Otero, Oscar; Alfonso, Carlos; Marcet, Ricardo; Sarracent, Jorge

    2016-09-01

    Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites

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

    PubMed

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

    2016-07-17

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

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

    SciTech Connect

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

    2007-03-15

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

  7. 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

  8. Identification of novel B cell epitopes within Toxoplasma gondii GRA1.

    PubMed

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

    2013-11-01

    Newly synthesized epitopes are one of the most promising antigens for the development of diagnostic kits and peptide vaccines. Very little is known about the B cell epitopes on GRA1 of Toxoplasma gondii, which are recognized by the humoral immune response in pigs. In this study, epitopes derived from GRA1 of T. gondii were identified using synthetic peptide techniques and bioinformatics. Three (PG10, PG13 and PG18) out of the eighteen peptides tested were recognized by all pig sera from different time points after infection, and the other peptides were recognized by select sera from various time points after infection. Our data indicate that many regions of GRA1, and in particular, the regions represented by the peptides PG10, PG13 and PG18, are involved in the pig antibody response. 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 epitope-based marker vaccines and diagnostic tools for toxoplasmosis. PMID:24090568

  9. 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

  10. 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

  11. Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts

    PubMed Central

    Maleki, Soheila J.; Teuber, Suzanne S.; Cheng, Hsiaopo; Chen, Deliang; Comstock, Sarah S.; Ruan, Sanbao; Schein, Catherine H.

    2011-01-01

    Background Cross reactivity between peanuts and tree nuts implies that similar IgE epitopes are present in their proteins. Objective To determine whether walnut sequences similar to known peanut IgE binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins (SDAP), react with IgE from sera of patients with allergy to walnut and/or peanut. Methods Patient sera were characterized by Western blotting for IgE-binding to nut protein extracts, and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive ELISA was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. Results Sequences from the vicilin walnut allergen Jug r 2 which had low PD values to epitopes of the peanut allergen Ara h 2, a 2s-albumin, bound IgE in sera from five patients who reacted to either walnut, peanut or both. A walnut epitope recognized by 6 patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. A predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. Conclusions Sequences with low PD value (<8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens. PMID:21883278

  12. 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

  13. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    PubMed Central

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  14. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

    PubMed

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia; Ahlborg, Niklas

    2016-09-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  15. 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

  16. 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

  17. 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

  18. The epitopes that cause cross-reactions between peanuts and tree nuts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many peanut allergic individuals also have allergies to tree nuts. Our previous work has shown that there are epitopes with different amino acid sequences, but similar physical and chemical properties are recognized by the same IgE molecule. Anti-Ara h 2 monoclonal antibodies were produced. They we...

  19. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  20. Preferential Nucleation during Polymorphic Transformations

    PubMed Central

    Sharma, H.; Sietsma, J.; Offerman, S. E.

    2016-01-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR’s) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR’s with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller – and therefore nucleation more probable - with increasing number of special OR’s. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material. PMID:27484579

  1. Preferential Nucleation during Polymorphic Transformations

    NASA Astrophysics Data System (ADS)

    Sharma, H.; Sietsma, J.; Offerman, S. E.

    2016-08-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR’s) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR’s with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller – and therefore nucleation more probable - with increasing number of special OR’s. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material.

  2. Preferential Nucleation during Polymorphic Transformations.

    PubMed

    Sharma, H; Sietsma, J; Offerman, S E

    2016-01-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR's) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR's with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller - and therefore nucleation more probable - with increasing number of special OR's. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material. PMID:27484579

  3. Multiplex localization of sequential peptide epitopes by use of a planar microbead chip.

    PubMed

    Schmidt, Carsten; Rödiger, Stefan; Gruner, Melanie; Moncsek, Anja; Stohwasser, Ralf; Hanack, Katja; Schierack, Peter; Schröder, Christian

    2016-02-18

    Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks. PMID:26826697

  4. Identification of HLA-A*11:01-restricted Mycobacterium tuberculosis CD8(+) T cell epitopes.

    PubMed

    Liu, Su-Dong; Su, Jin; Zhang, Shi-Meng; Dong, Hai-Ping; Wang, Hui; Luo, Wei; Wen, Qian; He, Jian-Chun; Yang, Xiao-Fan; Ma, Li

    2016-09-01

    New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette-Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug-resistant tuberculosis (MDR-TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8(+) T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8(+) T cell epitopes specific for MTB is essential for the design of peptide-based vaccines. To identify CD8(+) T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01-binding motifs. HLA-A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon-γ release and CD8(+) T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8(+) T cells from active pulmonary TB patients. In addition, a significant level of epitope-specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA-A*11:01 dextramer carrying the peptide Rv3130c194-204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8(+) T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB. PMID:27072810

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

    PubMed

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

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer. PMID:25483665

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

    PubMed Central

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

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer. PMID:25483665

  7. 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

  8. Recognizing Computational Science

    NASA Astrophysics Data System (ADS)

    Bland-Hawthorn, J.

    2006-08-01

    There are prestigious international awards that recognize the role of theory and experiment in science and mathematics, but there are no awards of a similar stature that explicitly recognize the role of computational science in a scientific field. In 1945, John von Neumann noted that "many branches of both pure and applied mathematics are in great need of computing instruments to break the present stalemate created by the failure of the purely analytical approach to nonlinear problems." In the past few decades, great strides in mathematics and in the applied sciences can be linked to computational science.

  9. 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

  10. 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

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

    PubMed Central

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

    1993-01-01

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

  12. Studies of epitope restriction on myeloperoxidase (MPO), an important antigen in systemic vasculitis

    PubMed Central

    SHORT, A K; LOCKWOOD, C M

    1997-01-01

    Anti-neutrophil cytoplasmic antibodies are important components of the inflammatory response in patients with systemic vasculitis. Their role in the pathogenesis of these conditions remains incompletely defined. Several antigens have been identified, and MPO is one of the most important. To gain more understanding of the immune mechanisms involved, we were keen to see if the antibody response to MPO was restricted, or whether there was a general loss of tolerance to the whole surface of the molecule. To study the epitopes we employed both ELISA and biosensor technology, and were able to demonstrate restriction both in the number and localization of the epitopes being recognized. PMID:9367412

  13. 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

  14. 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

  15. 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

  16. Conformational B-Cell Epitopes Prediction from Sequences Using Cost-Sensitive Ensemble Classifiers and Spatial Clustering

    PubMed Central

    Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use. PMID:25045691

  17. Antisera preparation and epitope mapping of a recombinant protein comprising three peptide fragments of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Li, Kun; Tang, Haiping; Xu, Wanxiang; Chen, Aijun; Shi, Qixian; Sun, Zhida; Wang, Liyan; Ni, Ya

    2015-10-01

    Antibodies targeting a single epitope of the cystic fibrosis transmembrane conductance regulator (CFTR) have been reported to influence the validity of immunological analyses; however, autoimmune mechanisms associated with CFTR epitopes are not well understood. In this study, antiserum raised against a multi-epitope recombinant protein composed of three peptide fragments of CFTR (r-CFTR-3P) was prepared and B cell epitope mapping of the protein was carried out using biosynthetic peptides. The r-CFTR-3P gene was cloned into the pSY621 expression plasmid and the protein was expressed in the BL21 strain of Escherichia coli. The rabbit r-CFTR-3P antiserum recognized the native CFTR antigen extracted from human sperm and the GST188 fusion peptides CFTR(25-36), CFTR(103-117), and CFTR(1387-1480) spanning different regions of CFTR. Four novel r-CFTR-3P B cell epitopes were identified: (29)RQRLEL(34), (104)RIIASY(109), (111)PDN(113), and (1447)VKLF(1450) of CFTR. Other proteins from various species shared sequence homology with the identified epitopes based on NCBI BLAST alignment. This study provides new tools for detecting CFTR protein and insight into the characteristics of minimal B cell epitopes of CFTR and associated immunological mechanisms. PMID:26087025

  18. Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

    PubMed

    Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use. PMID:25045691

  19. 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

  20. 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

  1. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

    NASA Astrophysics Data System (ADS)

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M.; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

  2. 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

  3. Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

    PubMed

    Grigera, P R; Keil, W; Wagner, R R

    1992-06-01

    Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which

  4. Information filtering via preferential diffusion

    NASA Astrophysics Data System (ADS)

    Lü, Linyuan; Liu, Weiping

    2011-06-01

    Recommender systems have shown great potential in addressing the information overload problem, namely helping users in finding interesting and relevant objects within a huge information space. Some physical dynamics, including the heat conduction process and mass or energy diffusion on networks, have recently found applications in personalized recommendation. Most of the previous studies focus overwhelmingly on recommendation accuracy as the only important factor, while overlooking the significance of diversity and novelty that indeed provide the vitality of the system. In this paper, we propose a recommendation algorithm based on the preferential diffusion process on a user-object bipartite network. Numerical analyses on two benchmark data sets, MovieLens and Netflix, indicate that our method outperforms the state-of-the-art methods. Specifically, it can not only provide more accurate recommendations, but also generate more diverse and novel recommendations by accurately recommending unpopular objects.

  5. Information filtering via preferential diffusion.

    PubMed

    Lü, Linyuan; Liu, Weiping

    2011-06-01

    Recommender systems have shown great potential in addressing the information overload problem, namely helping users in finding interesting and relevant objects within a huge information space. Some physical dynamics, including the heat conduction process and mass or energy diffusion on networks, have recently found applications in personalized recommendation. Most of the previous studies focus overwhelmingly on recommendation accuracy as the only important factor, while overlooking the significance of diversity and novelty that indeed provide the vitality of the system. In this paper, we propose a recommendation algorithm based on the preferential diffusion process on a user-object bipartite network. Numerical analyses on two benchmark data sets, MovieLens and Netflix, indicate that our method outperforms the state-of-the-art methods. Specifically, it can not only provide more accurate recommendations, but also generate more diverse and novel recommendations by accurately recommending unpopular objects. PMID:21797453

  6. On recognizing ignorance

    NASA Technical Reports Server (NTRS)

    Greene, Richard J.

    1988-01-01

    How an expert system reasons about its own ability to deal with a particular problem is studied. Ideally, an expert system ought to rapidly recognize that a particular problem is beyond its abilities and defer to another, perhaps human, expert. This capability is extremely important in domains where expert systems may control life critical processes such as air traffic control, medicine, strategic defense, and manned space exploration. The methods used by knowledge engineers to infuse an expert system with knowledge of its own limitations is surveyed. A computability theory is employed to analyze the general problem of meta-knowledge and to give insight into the efficacy of specific solutions.

  7. Preventing and Recognizing Embezzlement.

    PubMed

    Phairas, Debra

    2016-01-01

    It is estimated that approximately one in six physicians will be the victim of embezzlement at least once during his or her lifetime. This may be due to the trusting nature of physicians, a lack of business training about separating duties in transactions involving money, or employees' feeling overworked, underpaid, or underappreciated. The best protection against embezzlement is prevention. This article informs the reader of the steps to take to prevent stealing in the medical office, how to recognize if it is occurring, and how to obtain restitution or prosecution. PMID:27039633

  8. Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

    PubMed Central

    Turner, Kendrick B.; Zabetakis, Dan; Legler, Patricia; Goldman, Ellen R.; Anderson, George P.

    2014-01-01

    Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better. Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies. PMID:24949641

  9. 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

  10. Recognizing musical text

    NASA Astrophysics Data System (ADS)

    Clarke, Alastair T.; Brown, B. M.; Thorne, M. P.

    1993-08-01

    This paper reports on some recent developments in a software product that recognizes printed music notation. There are a number of computer systems available which assist in the task of printing music; however the full potential of these systems cannot be realized until the musical text has been entered into the computer. It is this problem that we address in this paper. The software we describe, which uses computationally inexpensive methods, is designed to analyze a music score, previously read by a flat bed scanner, and to extract the musical information that it contains. The paper discusses the methods used to recognize the musical text: these involve sampling the image at strategic points and using this information to estimate the musical symbol. It then discusses some hard problems that have been encountered during the course of the research; for example the recognition of chords and note clusters. It also reports on the progress that has been made in solving these problems and concludes with a discussion of work that needs to be undertaken over the next five years in order to transform this research prototype into a commercial product.

  11. 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

  12. Schistosoma mansoni shares a protective carbohydrate epitope with keyhole limpet hemocyanin

    PubMed Central

    1987-01-01

    The glycanic epitope of the 38,000 Mr Schistosoma mansoni schistosomula major immunogen defined by the IPLSm1 protective mAb was identified in the hemocyanin of the marine mollusc Megathura crenulata, better known as KLH. This antigenic community was exploited to investigate further the biological properties of this epitope. KLH was shown to strongly inhibit the binding of IPLSm1 mAb to its 38,000 Mr target antigen. Immunization of naive LOU rats with KLH elicited the production of anti- S. mansoni antibodies capable of immunoprecipitating the 38,000 Mr schistosomulum antigen. Antibodies to KLH mediated a marked eosinophil- dependent cytotoxicity and passively transferred immunity towards S. mansoni infection. Finally, rats immunized with KLH were significantly protected against a challenge with S. mansoni cercariae. The deglycosylation of KLH completely abolishes its immunological and functional KLH properties, indicating the participation of an oligosaccharidic epitope of the native KLH that is also recognized by the sera of S. mansoni-infected patients. These observations provide new opportunities of access to the well-defined structure of a glycanic epitope potentially available for the immunoprophylaxis and seroepidemiology of schistosomiasis, and a new approach to the isotypic response towards a well-chemically defined epitope. PMID:2434601

  13. NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences.

    PubMed

    Chuang, Gwo-Yu; Liou, David; Kwong, Peter D; Georgiev, Ivelin S

    2014-07-01

    Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

  14. Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    PubMed Central

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  15. Blister-inducing antibodies target multiple epitopes on collagen VII in mice.

    PubMed

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-09-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  16. Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization

    PubMed Central

    Newell, Evan W; Sigal, Natalia; Nair, Nitya; Kidd, Brian A; Greenberg, Harry B; Davis, Mark M

    2013-01-01

    It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide–MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide–MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines. PMID:23748502

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

    PubMed

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

    2009-05-01

    Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis. PMID:19363291

  18. 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

  19. Recognizing People in Motion.

    PubMed

    Yovel, Galit; O'Toole, Alice J

    2016-05-01

    Natural movements of the face and body, as well as voice, provide converging cues to a person's identity. To date, person recognition has been studied primarily with static images of faces. Face recognition, however, is part of a larger system, whose preeminent goal is to efficiently recognize dynamic familiar people in unconstrained environments. We present a comprehensive framework for understanding person recognition as it happens in the real world. In this framework, dynamic information plays the central role in binding multi-modal information from the face, body, and the voice to achieve robust and highly accurate recognition. The superior temporal sulcus (STS) integrates multisensory, dynamic information from the whole person for recognition, thereby complementing its role in social cognition. PMID:27016844

  20. Recognizing the Trends

    NASA Technical Reports Server (NTRS)

    Marley, Mark Scott

    2016-01-01

    Solar system planetary science has traditionally focused on understanding in depth individual planets. While there have been some efforts at synergy, most studies have focused on understanding the details of individual planets. Now that we are in the era of exoplanet science, with thousands of known planets and hundreds that have been characterized to varying degrees, the systematics of planetary science are becoming apparent. This also means that, for the first time, what had previously been seen as individual quirks of solar system planets are instead being recognized as part of the normal range of planetary behavior. In my talk I will consider a number of such characteristics and explain how we are now starting to understand their true context. In particular I will discuss the atmospheric composition, clouds, hazes, and winds of giant planets, trace gasses in the atmosphere of Venus, and the presence and absence of atmospheres on various terrestrial worlds.

  1. Recognizing outstanding achievements

    NASA Astrophysics Data System (ADS)

    Speiss, Fred

    One function of any professional society is to provide an objective, informed means for recognizing outstanding achievements in its field. In AGU's Ocean Sciences section we have a variety of means for carrying out this duty. They include recognition of outstanding student presentations at our meetings, dedication of special sessions, nomination of individuals to be fellows of the Union, invitations to present Sverdrup lectures, and recommendations for Macelwane Medals, the Ocean Sciences Award, and the Ewing Medal.Since the decision to bestow these awards requires initiative and judgement by members of our section in addition to a deserving individual, it seems appropriate to review the selection process for each and to urge you to identify those deserving of recognition.

  2. Detailed analyses of antibodies recognizing mitochondrial antigens suggest similar or identical mechanism for production of natural antibodies and natural autoantibodies.

    PubMed

    Czömpöly, Tamás; Olasz, Katalin; Nyárády, Zoltán; Simon, Diána; Bovári, Judit; Németh, Péter

    2008-06-01

    Because of their endosymbiotic evolutionary origin, proteins compartmentalized into mitochondria represent an interesting transition from prokaryotic foreign to essential self molecules. We investigated the presence of naturally occurring antibodies (nAbs) recognizing mitochondrial inner membrane enzymes. Epitope mapping analysis of a mitochondrial inner membrane enzyme, citrate synthase (CS) by synthetic overlapping peptides and phage display libraries using sera from healthy individuals and from patients having systemic autoimmune disease revealed CS recognizing nAbs with IgM isotype. We analyzed cross-reactive epitopes on human CS, bacterial CS, and various standard autoantigens. We have found that the fine epitope pattern on CS is different under physiological and pathological conditions. Moreover sera affinity purified on CS cross reacts with nucleosome antigen, which cross-reactivity could be mapped to a short epitope on human CS. These data indicate that in theory, nAbs "specific" for a given self antigen could fulfill the function of participating in innate defense mechanisms and at the same time recognize a target antigen in a systemic autoimmune disease. Thus, at the level of recognized epitopes there is a possible link between the innate like part and the adaptive-autoimmune arm of the humoral immune system. PMID:18558363

  3. Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3.

    PubMed

    Piesche, Matthias; Hildebrandt, York; Chapuy, Björn; Wulf, Gerald G; Trümper, Lorenz; Schroers, Roland

    2009-07-23

    Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegener's granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recognize antigens in the context of HLA-class-II-molecules, identification of HLA-class-II-associated peptide-epitopes from self-antigens such as PRTN3 is required. Here, we analyzed T-cell responses against proteinase 3 using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. We first screened a panel of ten epitope peptide candidates selected with the TEPITOPE program and found that nine out of ten peptides induced PRTN3 peptide-specific proliferation of T-cells with precursor frequencies of 0-1.1 x 10(-6). For one peptide-epitope, PRTN3(235), T-cell-clones were demonstrated to be capable of recognizing naturally processed protein antigen in a HLA-DR-restricted fashion. PRTN3(235)-specific T-cells could be stimulated from the blood of healthy individuals with multiple HLA-DR-genotypes. In summary, the identified PRTN3(235)-epitope can be used to study the role of CD4+ TH- and Treg-cells in immune responses against PRTN3 in leukemia patients and patients with Wegener's disease. PMID:19446593

  4. Do Karstic Unsaturated Zones Have the Fastest Preferential Flow?

    NASA Astrophysics Data System (ADS)

    Nimmo, J. R.; Perkins, K. S.

    2013-12-01

    There is strong evidence that unsaturated-zone travel times for preferential flow, unlike those for diffuse flow, mostly fall within a relatively small range, even for a wide variety of media and conditions [Nimmo, 2007, Water Resources Research]. We have calculated travel times for preferential flow observations published in the last seven years, finding a range of travel velocities with a greater maximum than was previously recognized. The instances of faster transport, however, are predominantly for karst or other materials in which water flow may strongly influence the creation and development of preferential flow paths. These findings motivate a hypothesis: in media where the matrix is soluble, erodible, or otherwise vulnerable to enlargement by flowing water, this flow acts to reduce flow impediments within a macropore network. This might be thought of as a sculpting process in which water carves its conduit into a smoother, larger, less constrictive shape, as discussed in connection with soil pipes [Jones, 2010, Hydrological Processes]. Known developmental processes of karst and epikarst are consistent with this hypothesis. Its acceptance would open doors to expanded use of optimality and thermodynamic principles to understand and predict preferential flow. It also could lead to new modes of hydraulic characterization of subsurface media with regard to unsaturated flow, which are much needed as the difficulty of measuring hydraulic properties of the unsaturated zone is a major barrier to the advance of hydrologic science. In practical terms, a new guideline may be justified: in unsaturated karst or other materials in which flowing water may enhance flowpaths, preferential transport rates in general may be several times faster than through media without such pore-developmental processes.

  5. A continuous peptide epitope reacting with pandemic influenza AH1N1 predicted by bioinformatic approaches.

    PubMed

    Carrillo-Vazquez, Jonathan P; Correa-Basurto, José; García-Machorro, Jazmin; Campos-Rodríguez, Rafael; Moreau, Violaine; Rosas-Trigueros, Jorge L; Reyes-López, Cesar A; Rojas-López, Marlon; Zamorano-Carrillo, Absalom

    2015-09-01

    Computational identification of potential epitopes with an immunogenic capacity challenges immunological research. Several methods show considerable success, and together with experimental studies, the efficiency of the algorithms to identify potential peptides with biological activity has improved. Herein, an epitope was designed by combining bioinformatics, docking, and molecular dynamics simulations. The hemagglutinin protein of the H1N1 influenza pandemic strain served as a template, owing to the interest of obtaining a scheme of immunization. Afterward, we performed enzyme-linked immunosorbent assay (ELISA) using the epitope to analyze if any antibodies in human sera before and after the influenza outbreak in 2009 recognize this peptide. Also, a plaque reduction neutralization test induced by virus-neutralizing antibodies and the IgG determination showed the biological activity of this computationally designed peptide. The results of the ELISAs demonstrated that the serum of both prepandemic and pandemic recognized the epitope. Moreover, the plaque reduction neutralization test evidenced the capacity of the designed peptide to neutralize influenza virus in Madin-Darby canine cells. PMID:25788327

  6. 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

  7. Thioreductase-Containing Epitopes Inhibit the Development of Type 1 Diabetes in the NOD Mouse Model

    PubMed Central

    Malek Abrahimians, Elin; Vander Elst, Luc; Carlier, Vincent A.; Saint-Remy, Jean-Marie

    2016-01-01

    Autoreactive CD4+ T cells recognizing islet-derived antigens play a primary role in type 1 diabetes. Specific suppression of such cells therefore represents a strategic target for the cure of the disease. We have developed a methodology by which CD4+ T cells acquire apoptosis-inducing properties on antigen-presenting cells after cognate recognition of natural sequence epitopes. We describe here that inclusion of a thiol-disulfide oxidoreductase (thioreductase) motif within the flanking residues of a single MHC class II-restricted GAD65 epitope induces GAD65-specific cytolytic CD4+ T cells (cCD4+ T). The latter, obtained either in vitro or by active immunization, acquire an effector memory phenotype and lyse APCs by a Fas–FasL interaction. Furthermore, cCD4+ T cells eliminate by apoptosis activated bystander CD4+ T cells recognizing alternative epitopes processed by the same APC. Active immunization with a GAD65 class II-restricted thioreductase-containing T cell epitope protects mice from diabetes and abrogates insulitis. Passive transfer of in vitro-elicited cCD4+ T cells establishes that such cells are efficient in suppressing autoimmunity. These findings provide strong evidence for a new vaccination strategy to prevent type 1 diabetes. PMID:26973647

  8. 15 CFR 700.14 - Preferential scheduling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE NATIONAL SECURITY INDUSTRIAL BASE REGULATIONS DEFENSE PRIORITIES AND ALLOCATIONS SYSTEM Industrial Priorities § 700.14 Preferential scheduling. (a)...

  9. How legumes recognize rhizobia

    PubMed Central

    Via, Virginia Dalla; Zanetti, María Eugenia; Blanco, Flavio

    2016-01-01

    ABSTRACT Legume plants have developed the capacity to establish symbiotic interactions with soil bacteria (known as rhizobia) that can convert N2 to molecular forms that are incorporated into the plant metabolism. The first step of this relationship is the recognition of bacteria by the plant, which allows to distinguish potentially harmful species from symbiotic partners. The main molecular determinant of this symbiotic interaction is the Nod Factor, a diffusible lipochitooligosaccharide molecule produced by rhizobia and perceived by LysM receptor kinases; however, other important molecules involved in the specific recognition have emerged over the years. Secreted exopolysaccharides and the lipopolysaccharides present in the bacterial cell wall have been proposed to act as signaling molecules, triggering the expression of specific genes related to the symbiotic process. In this review we will briefly discuss how transcriptomic analysis are helping to understand how multiple signaling pathways, triggered by the perception of different molecules produced by rhizobia, control the genetic programs of root nodule organogenesis and bacterial infection. This knowledge can help to understand how legumes have evolved to recognize and establish complex ecological relationships with particular species and strains of rhizobia, adjusting gene expression in response to identity determinants of bacteria. PMID:26636731

  10. T Cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr Virus

    PubMed Central

    Rist, Melissa J.; Neller, Michelle A.; Burrows, Jacqueline M.

    2014-01-01

    ABSTRACT Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating “immune blind spots” through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by

  11. 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

  12. 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

  13. Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP

    PubMed Central

    van Bergen, Jeroen; Schoenberger, Stephen P.; Verreck, Frank; Amons, Reinout; Offringa, Rienk; Koning, Frits

    1997-01-01

    The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized. PMID:9207120

  14. 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

  15. 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.

  16. Epitope mapping for monoclonal antibodies recognizing tuber necrotic isolates of Potato virus Y

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato virus Y (PVY) is an important viral pathogen of potato responsible for reducing tuber yield and quality across the globe. The PVYN and PVYNTN strains, the latter of which induces potato tuber necrotic ringspot disease (PTNRD), are regulated for international potato trade, and have been routin...

  17. 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

  18. 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

  19. 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

  20. Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

    PubMed Central

    Comerford, S A; McCance, D J; Dougan, G; Tite, J P

    1991-01-01

    There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant. Images PMID:1714516

  1. Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri.

    PubMed

    Phalipon, A; Arondel, J; Nato, F; Rouyre, S; Mazie, J C; Sansonetti, P J

    1992-05-01

    Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection. PMID:1373401

  2. 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

  3. Analysis of the immune response of a new malaria vaccine based on the modification of cryptic epitopes.

    PubMed

    Shen, Yan; Wang, Jun; Huang, Yuxiao; Liang, Jiao; Liu, Xuewu; Wu, Dudu; Jiang, He; Zhao, Ya; Li, Yinghui

    2016-05-01

    Malaria is a severe, life-threatening infectious disease that endangers human health. However, there are no vaccines or immune strategy of vaccines succeeding in both erythrocytic and pre-erythrocytic stage. During the liver stage of the Plasmodium life cycle, sporozoites invade the host liver cells. The sporozoites, then, induce a cellular immune response via the major histocompatibility complex (MHC) molecules on their surfaces. The cytotoxic T lymphocytes (CTLs) then recognize the corresponding antigen-MHC complex on the surfaces of these infected liver cells and kill them. However, dominant epitopes with high MHC affinity are prone to mutation due to immune selection pressure. CTLs evoked by the original dominant epitopes cannot recognize the mutated epitopes, leading to immune evasion. In this study, we have modified the cryptic epitopes of different antigens in the sporozoite and liver stages of Plasmodium falciparum to increase their immunogenicity without changing T cell antigen receptor (TCR)-peptide binding specificity. In addition, we have also added an important erythrocytic phase protective antigen, named apical membrane antigen 1 (AMA-1), to this process with the goal of constructing a complex multi-stage, multi-epitope recombinant DNA vaccine against P. falciparum. The vaccine was tested in HHD-2 mice. The method involved multiple stages of the P. falciparum life cycle as well as elucidation both humoral and cellular immunity. The conclusion drawn from the study was that the vaccine might provide an important theoretical and practical basis for generating effective preventative or therapeutic vaccine against P. falciparum. PMID:26833322

  4. The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

    PubMed Central

    Polymenidou, Magdalini; Moos, Rita; Scott, Mike; Sigurdson, Christina; Shi, Yong-zhong; Yajima, Bill; Hafner-Bratkovič, Iva; Jerala, Roman; Hornemann, Simone; Wuthrich, Kurt; Bellon, Anne; Vey, Martin; Garen, Graciela; James, Michael N. G.; Kav, Nat; Aguzzi, Adriano

    2008-01-01

    PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays. PMID:19060956

  5. 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

  6. Glycocalyx on rabbit intestinal M cells displays carbohydrate epitopes from Muc2.

    PubMed

    Lelouard, H; Reggio, H; Roy, C; Sahuquet, A; Mangeat, P; Montcourrier, P

    2001-02-01

    It is essential to investigate the apical surface properties of both M cells and dome enterocytes to understand the mechanisms involved in the binding of pathogens to M cells. In rabbit appendix tissue, monoclonal antibodies (MAbs) highlight differences between M cells (MAb 58) and dome enterocytes (MAb 214). Such antibodies ultimately recognized intestinal mucin-related epitopes. To further characterize these differences, the labeling patterns obtained with these MAbs were compared to those obtained with other antibodies to intestinal mucins on dissected domes from all gut-associated lymphoid tissues. A glycoprotein recognized by MAb 58 was purified on a CsCl isopycnic density gradient and microsequenced, and its mRNA expression was localized by in situ hybridization. It was identified as the rabbit homologue of human Muc2, i.e., the major mucin secreted in intestine tissue. Two other Muc2 carbohydrate epitopes were also expressed on M cells, although Muc2 mRNA was not detected. All results indicated that M cells express, on their apical membrane, glycoconjugates bearing at least three glycosidic epitopes from Muc2. MAb 214 and MAb 6G2, which recognized a partially characterized mucin expressed on dome enterocytes, were negative markers for M cells in rabbit gut-associated lymphoid tissues. We propose that the presence, on the surface of M cells, of carbohydrates also expressed on Muc2, together with the absence of an enterocyte-associated mucin, could favor pathogen attachment and accessibility to the M-cell luminal membrane. PMID:11160003

  7. Glycocalyx on Rabbit Intestinal M Cells Displays Carbohydrate Epitopes from Muc2

    PubMed Central

    Lelouard, Hugues; Reggio, Hubert; Roy, Christian; Sahuquet, Alain; Mangeat, Paul; Montcourrier, Philippe

    2001-01-01

    It is essential to investigate the apical surface properties of both M cells and dome enterocytes to understand the mechanisms involved in the binding of pathogens to M cells. In rabbit appendix tissue, monoclonal antibodies (MAbs) highlight differences between M cells (MAb 58) and dome enterocytes (MAb 214). Such antibodies ultimately recognized intestinal mucin-related epitopes. To further characterize these differences, the labeling patterns obtained with these MAbs were compared to those obtained with other antibodies to intestinal mucins on dissected domes from all gut-associated lymphoid tissues. A glycoprotein recognized by MAb 58 was purified on a CsCl isopycnic density gradient and microsequenced, and its mRNA expression was localized by in situ hybridization. It was identified as the rabbit homologue of human Muc2, i.e., the major mucin secreted in intestine tissue. Two other Muc2 carbohydrate epitopes were also expressed on M cells, although Muc2 mRNA was not detected. All results indicated that M cells express, on their apical membrane, glycoconjugates bearing at least three glycosidic epitopes from Muc2. MAb 214 and MAb 6G2, which recognized a partially characterized mucin expressed on dome enterocytes, were negative markers for M cells in rabbit gut-associated lymphoid tissues. We propose that the presence, on the surface of M cells, of carbohydrates also expressed on Muc2, together with the absence of an enterocyte-associated mucin, could favor pathogen attachment and accessibility to the M-cell luminal membrane. PMID:11160003

  8. Mapping of multiple HLA class II-restricted T-cell epitopes of the mycobacterial 70-kilodalton heat shock protein.

    PubMed Central

    Oftung, F; Geluk, A; Lundin, K E; Meloen, R H; Thole, J E; Mustafa, A S; Ottenhoff, T H

    1994-01-01

    By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules. PMID:7525484

  9. 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

  10. 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

  11. Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

    PubMed Central

    Darji, A; Niebuhr, K; Hense, M; Wehland, J; Chakraborty, T; Weiss, S

    1996-01-01

    Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity. PMID:8675351

  12. Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury1

    PubMed Central

    Kulik, Liudmila; Fleming, Sherry D.; Moratz, Chantal; Reuter, Jason W.; Novikov, Aleksey; Chen, Kuan; Andrews, Kathy A.; Markaryan, Adam; Quigg, Richard J.; Silverman, Gregg J.; Tsokos, George C.; Holers, V. Michael

    2010-01-01

    Intestinal ischemia-reperfusion (IR)3 injury is initiated when natural IgM antibodies recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild type C57BL/6 mice. We identified a novel IgM monoclonal antibody (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1−/− mice. Monoclonal Ab B4 was found to specifically recognize mouse annexin IV. Pre-injection of recombinant annexin IV blocked IR injury in wild type C57BL/6 mice, demonstrating the requirement for recognition of this protein in order to develop IR injury in the context of a complex natural antibody repertoire. Humans were also found to exhibit IgM natural antibodies that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target antigen that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury. PMID:19380783

  13. 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

  14. Dissociation between Epitope Hierarchy and Immunoprevalence in CD8 Responses to Vaccinia Virus Western Reserve1

    PubMed Central

    Oseroff, Carla; Peters, Bjoern; Pasquetto, Valerie; Moutaftsi, Magdalini; Sidney, John; Panchanathan, Vijay; Tscharke, David C.; Maillere, Bernard; Grey, Howard; Sette, Alessandro

    2008-01-01

    Understanding immunity to vaccinia virus (VACV) is important for the development of safer vaccines for smallpox- and poxvirus-vectored recombinant vaccines. VACV is also emerging as an outstanding model for studying CD8+ T cell immunodominance because of the large number of CD8+ T cell epitopes known for this virus in both mice and humans. In this study, we characterize the CD8+ T cell response in vaccinated BALB/c mice by a genome-wide mapping approach. Responses to each of 54 newly identified H-2d-restricted T cell epitopes could be detected after i.p. and dermal vaccination routes. Analysis of these new epitopes in the context of those already known for VACV in mice and humans revealed two important findings. First, CD8+ T cell epitopes are not randomly distributed across the VACV proteome, with some proteins being poorly or nonimmunogenic, while others are immunoprevalent, being frequently recognized across diverse MHC haplotypes. Second, some proteins constituted the major targets of the immune response by a specific haplotype as they recruited the majority of the specific CD8+ T cells but these proteins did not correspond to the immunoprevalent Ags. Thus, we found a dissociation between immunoprevalence and immunodominance, implying that different sets of rules govern these two phenomena. Together, these findings have clear implications for the design of CD8+ T cell subunit vaccines and in particular raise the exciting prospect of being able to choose subunits without reference to MHC restriction. PMID:18490718

  15. Identification of a promiscuous HLA DR-restricted T-cell epitope derived from the inhibitor of apoptosis protein survivin.

    PubMed

    Piesche, Matthias; Hildebrandt, York; Zettl, Florian; Chapuy, Björn; Schmitz, Marc; Wulf, Gerald; Trümper, Lorenz; Schroers, Roland

    2007-07-01

    The inhibitor of apoptosis protein survivin is a promising tumor-associated antigen specifically recognized by CD8+ cytotoxic effector T-lymphocytes (CTL). To improve current vaccines that aim to induce survivin-specific CTL, it is necessary to study the role of CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Because both TH and Treg cells recognize antigens in the context of HLA-class II molecules, identification of HLA class II-associated peptide epitopes from survivin is required. Here, we analyzed T-cell responses against survivin using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. Six peptides were shown to induce CD4+ T-cell responses, restricted by HLA-DR molecules. For one peptide epitope, SVN10, T-cell clones were demonstrated to be capable of recognizing naturally processed antigen. SVN10-specific T cells could be stimulated from the blood of healthy individuals and cancer patients with multiple HLA-DR genotypes. Thus the identified SVN10 epitope can be used to study the role of CD4+ TH and Treg cells in immune responses and possibly be included in a multivalent peptide vaccine against survivin. PMID:17584578

  16. Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tpp15) with synthetic peptides.

    PubMed Central

    Baughn, R E; Demecs, M; Taber, L H; Musher, D M

    1996-01-01

    The antigenicity of the 15-kDa lipoprotein of Treponema pallidum (Tpp15 or TpN15) was comprehensively evaluated in epitope-scanning studies with overlapping deca- and octapeptides and polygonal rabbit and human infant immunoglobulins (Igs) and antisera. This approach enabled us to identify potentially important regions and to determine the optimal dilutions of Igs or antisera for use in further studies. IgM and IgG from both species were capable of recognizing multiple, continuous epitopes. A total of 13 peptides, principally clustered in the central regions of the protein, were recognized by all syphilitic sera and Ig fractions. On the basis of window analyses, frequency profiles, and alanine substitution studies, five heptapeptides were selected for mimetic studies. Two of these five immunodominant, continuous epitopes initially appeared to be species specific; however, antisera elicited against mimetics of all five epitopes were polyspecific, recognizing similar motifs on several other treponemal proteins, including those of avirulent organisms. The only mimetic which yielded positive reactions with infant IgM and syphilitic sera in the absence of cross-reactions with rabbit antisera to avirulent treponemes was the variant of the VMYASSG motif. These findings are relevant to the development of simple, inexpensive assays for the serodiagnosis of active syphilis. PMID:8698467

  17. 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

  18. 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

  19. 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

  20. Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

    PubMed

    Sparer, T E; Wynn, S G; Clark, D J; Kaplan, J M; Cardoza, L M; Wadsworth, S C; Smith, A E; Gooding, L R

    1997-03-01

    Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population. PMID:9032363

  1. 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

  2. The epitope analysis of an antibody specifically against Vibrio cholerae O1 Ogawa by phage library study.

    PubMed

    Cheng, Shiliang; Lin, Zhen; Liu, Xinfeng; Zheng, Wen; Lu, Gang; Tu, Zhiguang; Zhang, Jun; Zheng, Jian; Yu, Xiaolin

    2015-10-01

    To prevent epidemic and pandemic cholera disease, an indispensible approach is to develop cholera vaccines based on comprehensive epitope information of this pathogen. This study aimed to utilize our previously raised monoclonal antibody IXiao3G6, which can recognize an epitope in lipopolysaccharide (LPS) sites of Ogawa, to identify mimetic peptides, which may represent Ogawa LPS's epitope information. A phage display library screening using IXiao3G6 antibody resulted in identification of a mimic peptide (MP) with high avidity. A recombinant protein, containing one cholera toxin subunit B (CTB) and two MP repeats (CTB-(MP)2), was subsequently constructed and investigated for its immunological characteristics. The findings collectively demonstrated that the MP presenting phages and CTB-(MP)2 recombinant protein were both capable of inhibiting the interaction between IXiao3G6 and Ogawa/Ogawa LPS specifically in a dose-dependent manner. PMID:26172085

  3. Establishment of Schistosoma japonicum calpain-specific mouse T cell hybridomas and identification of a T cell epitope that stimulates IFNgamma production.

    PubMed

    Osada, Yoshio; Kumagai, Takashi; Hato, Mariko; Suzuki, Takashi; El-Malky, Mohamed; Asahi, Hiroko; Kanazawa, Tamotsu; Ohta, Nobuo

    2005-04-15

    Calpain is a calcium-dependent cystein protease, and the homologues of schistosome are known as one of vaccine candidate molecules against schistosomiasis. Here, we established two IL-2 producing T cell hybridoma cell lines specific for Schistosoma japonicum calpain, to identify T cell epitope(s) on the molecule. Overlapping 15mer oligopeptides of calpain were synthesized and tested for their stimulatory abilities to the hybridomas. As a result, epitopes recognized by the two hybridoma lines were the same: EQLKIYAQRC. Spleen cells from calpain multiple antigenic peptide (MAP)-immunized BALB/c mice produced IFNgamma upon stimulation with MAP or soluble worm antigen preparation (SWAP). The identification of the T cell epitope to stimulate Th1 response will contribute to the proper design of synthetic vaccines, evaluation of their protective potentials and elucidation of protective mechanisms in murine experimental schistosomiasis. PMID:15780729

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

    PubMed

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

    1990-06-01

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

  5. A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV

    PubMed Central

    Wen, Xiaolin; Pickens, Jennifer; Mousa, Jarrod J.; Leser, George P.; Lamb, Robert A.; Crowe, James E.; Jardetzky, Theodore S.

    2016-01-01

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections. PMID:27224013

  6. Identification of autoantigens recognized by the 2F5 and 4E10 broadly neutralizing HIV-1 antibodies

    PubMed Central

    Yang, Guang; Holl, T. Matt; Liu, Yang; Li, Yi; Lu, Xiaozhi; Nicely, Nathan I.; Kepler, Thomas B.; Alam, S. Munir; Liao, Hua-Xin; Cain, Derek W.; Spicer, Leonard; VandeBerg, John L.; Haynes, Barton F.

    2013-01-01

    Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance because mice expressing the VH and VL regions of 2F5 have a block in B cell development that is characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. We identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes an epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but they retain the SF3B3 4E10 epitope. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motifs shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. PMID:23359068

  7. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

    PubMed Central

    Meinl, E; Weber, F; Drexler, K; Morelle, C; Ott, M; Saruhan-Direskeneli, G; Goebels, N; Ertl, B; Jechart, G; Giegerich, G

    1993-01-01

    The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an

  8. 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

  9. 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

  10. 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

  11. 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

  12. Preferential sampling in veterinary parasitological surveillance.

    PubMed

    Cecconi, Lorenzo; Biggeri, Annibale; Grisotto, Laura; Berrocal, Veronica; Rinaldi, Laura; Musella, Vincenzo; Cringoli, Giuseppe; Catelan, Dolores

    2016-01-01

    In parasitological surveillance of livestock, prevalence surveys are conducted on a sample of farms using several sampling designs. For example, opportunistic surveys or informative sampling designs are very common. Preferential sampling refers to any situation in which the spatial process and the sampling locations are not independent. Most examples of preferential sampling in the spatial statistics literature are in environmental statistics with focus on pollutant monitors, and it has been shown that, if preferential sampling is present and is not accounted for in the statistical modelling and data analysis, statistical inference can be misleading. In this paper, working in the context of veterinary parasitology, we propose and use geostatistical models to predict the continuous and spatially-varying risk of a parasite infection. Specifically, breaking with the common practice in veterinary parasitological surveillance to ignore preferential sampling even though informative or opportunistic samples are very common, we specify a two-stage hierarchical Bayesian model that adjusts for preferential sampling and we apply it to data on Fasciola hepatica infection in sheep farms in Campania region (Southern Italy) in the years 2013-2014. PMID:27087037

  13. On preferential flow and its measurement

    SciTech Connect

    Luxmoore, R.J.

    1991-01-01

    Preferential flow is a useful generic term for describing the process whereby water movement through a porous medium follows favored routes bypassing other parts of the medium. This term does not give any indication of the pore scales involved. Sometimes macropore flow is used to describe preferential flow and this term implies that large pores of some sort are conductive. There is no consensus definition of what constitutes a macropore so one needs to carefully determine what is meant when that term is used. The main focus of this report is on the measurement and characterization of preferential flow through structured soils, however, preferred path flow also occurs in sandy soils. Fingering flow in soils, a result of wetting front instability, is a third type of preferential flow that occurs in porous media with more or less random pore arrangement. There may not be any physically defined channels in the soil to account for this type of flow. A larger scale flow described as funnel flow by Kung et al. (1990) results from profile heterogeneity. Low permeability layers or coarse lenses in a profile may restrict vertical drainage redirecting flow laterally through specific regions of the profile (like a funnel). Water repellency can also be a factor in the development of preferential flow. 34 refs.

  14. Discovering Preferential Patterns in Sectoral Trade Networks.

    PubMed

    Cingolani, Isabella; Piccardi, Carlo; Tajoli, Lucia

    2015-01-01

    We analyze the patterns of import/export bilateral relations, with the aim of assessing the relevance and shape of "preferentiality" in countries' trade decisions. Preferentiality here is defined as the tendency to concentrate trade on one or few partners. With this purpose, we adopt a systemic approach through the use of the tools of complex network analysis. In particular, we apply a pattern detection approach based on community and pseudocommunity analysis, in order to highlight the groups of countries within which most of members' trade occur. The method is applied to two intra-industry trade networks consisting of 221 countries, relative to the low-tech "Textiles and Textile Articles" and the high-tech "Electronics" sectors for the year 2006, to look at the structure of world trade before the start of the international financial crisis. It turns out that the two networks display some similarities and some differences in preferential trade patterns: they both include few significant communities that define narrow sets of countries trading with each other as preferential destinations markets or supply sources, and they are characterized by the presence of similar hierarchical structures, led by the largest economies. But there are also distinctive features due to the characteristics of the industries examined, in which the organization of production and the destination markets are different. Overall, the extent of preferentiality and partner selection at the sector level confirm the relevance of international trade costs still today, inducing countries to seek the highest efficiency in their trade patterns. PMID:26485163

  15. A generalized theory of preferential linking

    NASA Astrophysics Data System (ADS)

    Hu, Haibo; Guo, Jinli; Liu, Xuan; Wang, Xiaofan

    2014-12-01

    There are diverse mechanisms driving the evolution of social networks. A key open question dealing with understanding their evolution is: How do various preferential linking mechanisms produce networks with different features? In this paper we first empirically study preferential linking phenomena in an evolving online social network, find and validate the linear preference. We propose an analyzable model which captures the real growth process of the network and reveals the underlying mechanism dominating its evolution. Furthermore based on preferential linking we propose a generalized model reproducing the evolution of online social networks, and present unified analytical results describing network characteristics for 27 preference scenarios. We study the mathematical structure of degree distributions and find that within the framework of preferential linking analytical degree distributions can only be the combinations of finite kinds of functions which are related to rational, logarithmic and inverse tangent functions, and extremely complex network structure will emerge even for very simple sublinear preferential linking. This work not only provides a verifiable origin for the emergence of various network characteristics in social networks, but bridges the micro individuals' behaviors and the global organization of social networks.

  16. Alzheimer's neurofibrillary tangles contain unique epitopes and epitopes in common with the heat-stable microtubule associated proteins tau and MAP2.

    PubMed Central

    Yen, S. H.; Dickson, D. W.; Crowe, A.; Butler, M.; Shelanski, M. L.

    1987-01-01

    Ten monoclonal antibodies raised against Alzheimer's neurofibrillary tangles (ANTs) were characterized for reactivity with heat-stable microtubule fractions from bovine and human brain. Five of the antibodies showed very little reaction, but the other five reacted strongly with heat-stable microtubule associated proteins (MAPs). The proteins recognized by these antibodies have estimated molecular weights similar to those of known heat-stable MAPs, tau (52-68 kd) and MAP2 (200-250 kd). That the proteins are indeed tau and MAP2 is demonstrated by reaction of electroblotted proteins with antibodies raised in mouse and guinea pig against bovine brain tau and MAP2. One anti-ANT antibody reacts only with tau, two bind strongly to tau and weakly to MAP2, one recognizes both tau and MAP2 equally well, and one primarily stains MAP2. Extraction of ANT with 2% SDS does not remove tau or MAP2 epitopes from ANT, indicating that epitopes shared with heat-stable MAPs are integral components of ANT. The existence of tau epitopes in ANT is also demonstrated by immunoblotting of ANT-enriched fractions with anti-tau antibodies. Most of the material recognized by anti-tau antibodies in ANT-enriched fractions is present in large molecules excluded by 3% polyacrylamide gel upon electrophoresis. Anti-tau antibodies immunostain ANT in immunofluorescence and immunoperoxidase studies. The immunostaining can be blocked by absorption of anti-tau antibodies with purified tau proteins from bovine brain. Not all ANTs in any given tissue section or isolated Alzheimer perikarial preparations, however, are stained by anti-tau antibodies. These results are consistent with previous studies that have demonstrated heterogeneity of ANTs. Whether this heterogeneity is due to biochemical modification of MAPs or absence of MAPs in some ANTs is unknown. The significance of what appear to be shared epitopes recognized by monoclonal antibodies in tau and MAP2, and the implications this may have on the

  17. 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

  18. 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

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

    PubMed

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

    2016-10-01

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

  20. Heligmosomoides polygyrus elicits a dominant nonprotective antibody response directed against restricted glycan and peptide epitopes.

    PubMed

    Hewitson, James P; Filbey, Kara J; Grainger, John R; Dowle, Adam A; Pearson, Mark; Murray, Janice; Harcus, Yvonne; Maizels, Rick M

    2011-11-01

    Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory-secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity. PMID:21964031

  1. Structural Analysis of a Protective Epitope of the Francisella tularensis O-Polysaccharide†

    PubMed Central

    Rynkiewicz, Michael J.; Lu, Zhaohua; Hui, Julia H.; Sharon, Jacqueline; Seaton, Barbara A.

    2012-01-01

    Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, is considered a biothreat due to its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS. To further localize the Ab52 epitope and understand the molecular interactions between the antibody and the saccharide, we now solved the X-ray crystal structure of the Fab fragment of Ab52 and derived an antibody-antigen complex using molecular docking. The docked complex, refined through energy minimization, reveals an antigen binding site in the shape of a large canyon with a central pocket that accommodates a V-shaped epitope consisting of six sugar residues, α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN(1→3)-β-D-QuipNAc(1→2)-β-D-Quip4NFm(1→4)-α-D-GalpNAcAN(1→4)-α-D-GalpNAcAN. These results inform the development of vaccines and immunotherapeutic/immunoprophylactic antibodies against Ft by suggesting a desired topology for antibody binding to internal epitopes of Ft LPS. This is the first report of an X-ray crystal structure of a monoclonal antibody that targets a protective Ft B cell epitope. PMID:22747335

  2. Structure-Based Design of a Protein Immunogen that Displays an HIV-1 gp41 Neutralizing Epitope

    SciTech Connect

    Stanfield, Robyn L.; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S.; Zwick, Michael B.; Wilson, Ian A.

    2012-06-27

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K{sub d} of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  3. Epitope specificity of spontaneous and induced thyroglobulin autoantibodies in the rat.

    PubMed Central

    De Assis-Paiva, H J; Champion, B; Rayner, D C; Colle, E; Bone, A; Roitt, I M; Cooke, A

    1988-01-01

    We have investigated the epitope specificities of rat thyroglobulin (Tg) autoantibodies arising either spontaneously in BB hybrid and BB rats or following induction in normal rats with thyroglobulin and adjuvant. Using a panel of thyroglobulins from different animal species it was possible to identify three different patterns of reactivity. These were: 1) recognition of all species of thyroglobulin; (2) recognition restricted to rat and mouse thyroglobulins and 3) recognition biased towards dog, rat and mouse thyroglobulins. Furthermore, using human thyroglobulin manifesting different levels of iodination, it was possible to show that sera with recognition pattern 1 recognized the iodination site of thyroglobulin and that this was inhibitable by thyroxine. Taken together these data provide evidence of restricted epitope recognition by Tg autoantibodies in the rat. PMID:2464449

  4. Diversity in CD8+ T Cell Function and Epitope Breadth Among Persons with Genital Herpes

    PubMed Central

    Laing, Kerry J.; Magaret, Amalia S.; Mueller, Dawn E.; Zhao, Lin; Johnston, Christine; De Rosa, Stephen C.; Koelle, David M.; Wald, Anna

    2010-01-01

    CD8+ T cells are known to be important in clearing herpes simplex virus (HSV) infections. However, investigating the specific antiviral mechanisms employed by HSV-2-specific T cell populations is limited by a lack of reagents such as CD8+ T cell epitopes and specific tetramers. Using a combination of intracellular cytokine staining flow cytometry and ELISpot methods, we functionally characterized peripheral HSV-2-specific CD8+ T cells from peripheral blood mononuclear cell (PBMC) that recognize 14 selected HSV-2 open-reading frames (ORFs) from 55 HSV-2 seropositive persons; within these ORFs, we subsequently identified more than 20 unique CD8+ T cell epitopes. CD8+ T cells to HSV-2 exhibited significant heterogeneity in their functional characteristics, proliferation, production of inflammatory cytokines, and potential to degranulate ex vivo. The diversity in T cell response in these ex vivo assessments offers the potential of defining immune correlates of HSV-2 reactivation in humans. PMID:20635156

  5. 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

  6. 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

  7. 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

  8. Reverse preferential spread in complex networks

    NASA Astrophysics Data System (ADS)

    Toyoizumi, Hiroshi; Tani, Seiichi; Miyoshi, Naoto; Okamoto, Yoshio

    2012-08-01

    Large-degree nodes may have a larger influence on the network, but they can be bottlenecks for spreading information since spreading attempts tend to concentrate on these nodes and become redundant. We discuss that the reverse preferential spread (distributing information inversely proportional to the degree of the receiving node) has an advantage over other spread mechanisms. In large uncorrelated networks, we show that the mean number of nodes that receive information under the reverse preferential spread is an upper bound among any other weight-based spread mechanisms, and this upper bound is indeed a logistic growth independent of the degree distribution.

  9. Biochemical, Biophysical and IgE-Epitope Characterization of the Wheat Food Allergen, Tri a 37

    PubMed Central

    Pahr, Sandra; Selb, Regina; Weber, Milena; Focke-Tejkl, Margarete; Hofer, Gerhard; Dordić, Andela; Keller, Walter; Papadopoulos, Nikolaos G.; Giavi, Stavroula; Mäkelä, Mika; Pelkonen, Anna; Niederberger, Verena; Vrtala, Susanne; Valenta, Rudolf

    2014-01-01

    Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptid were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential Ig

  10. Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

    PubMed

    Pahr, Sandra; Selb, Regina; Weber, Milena; Focke-Tejkl, Margarete; Hofer, Gerhard; Dordić, Andela; Keller, Walter; Papadopoulos, Nikolaos G; Giavi, Stavroula; Mäkelä, Mika; Pelkonen, Anna; Niederberger, Verena; Vrtala, Susanne; Valenta, Rudolf

    2014-01-01

    Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential Ig

  11. Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

    PubMed Central

    Schaefer, Deborah A.; Auerbach-Dixon, Beth A.; Riggs, Michael W.

    2000-01-01

    The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis. PMID:10768951

  12. 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

  13. Recognizing apathy in Alzheimer's disease.

    PubMed

    Lerner, Alan J; Strauss, Milton; Sami, Susie A

    2007-11-01

    Apathy has been increasingly recognized as a neuropsychiatric symptom in many neurologic disorders. In this paper, we review the clinical features of apathy in Alzheimer's disease. We also review screening, the differential diagnosis including depression, medical illnesses, and mild cognitive impairment, and treating modalities and issues. It must also be recognized that apathy per se almost never occurs as an isolated syndrome, so it must be viewed in the context of an individual's entire behavioral and cognitive status. PMID:17999565

  14. 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

  15. Antibodies recognizing a variety of different structural motifs on meningococcal Lip antigen fail to demonstrate bactericidal activity.

    PubMed

    Tinsley, C R; Virji, M; Heckels, J E

    1992-11-01

    The neisserial Lip antigen is a conserved antigen associated with the pathogenic Neisseria species, and is composed of multiple repeats of a consensus pentapeptide. A series of monoclonal antibodies reacting with meningococcal Lip antigen were subjected to epitope mapping, using solid-phase synthetic peptides based on the consensus repeat sequence. The antibodies were found to recognize different continuous epitopes based on the consensus sequence. One monoclonal antibody was utilized in affinity chromatography to obtain purified Lip antigen and the antigen was used for immunization of mice. The resulting antisera did not recognize Lip antigen on Western blots but reacted specifically with Lip antigen in immune precipitation experiments, indicating that the predominant polyclonal immune response was directed against conformational epitopes. Despite the diversity of both continuous and conformational epitopes recognized by the antibodies produced, none of the antibodies demonstrated the ability to promote complement-mediated bactericidal activity. Thus despite its initial apparent promise as a potential vaccine candidate the case for the inclusion of Lip antigen in vaccine formulation cannot be supported at present. PMID:1282535

  16. A generalized distance function for preferential choices.

    PubMed

    Berkowitsch, Nicolas A J; Scheibehenne, Benjamin; Rieskamp, Jörg; Matthäus, Max

    2015-05-01

    Many cognitive theories of judgement and decision making assume that choice options are evaluated relative to other available options. The extent to which the preference for one option is influenced by other available options will often depend on how similar the options are to each other, where similarity is assumed to be a decreasing function of the distance between options. We examine how the distance between preferential options that are described on multiple attributes can be determined. Previous distance functions do not take into account that attributes differ in their subjective importance, are limited to two attributes, or neglect the preferential relationship between the options. To measure the distance between preferential options it is necessary to take the subjective preferences of the decision maker into account. Accordingly, the multi-attribute space that defines the relationship between options can be stretched or shrunk relative to the attention or importance that a person gives to different attributes describing the options. Here, we propose a generalized distance function for preferential choices that takes subjective attribute importance into account and allows for individual differences according to such subjective preferences. Using a hands-on example, we illustrate the application of the function and compare it to previous distance measures. We conclude with a discussion of the suitability and limitations of the proposed distance function. PMID:25677976

  17. Preferential attachment in randomly grown networks

    NASA Astrophysics Data System (ADS)

    Weaver, Iain S.

    2015-12-01

    We reintroduce the model of Callaway et al. (2001) as a special case of a more general model for random network growth. Vertices are added to the graph at a rate of 1, while edges are introduced at rate δ. Rather than edges being introduced at random, we allow for a degree of preferential attachment with a linear attachment kernel, parametrised by m. The original model is recovered in the limit of no preferential attachment, m → ∞. As expected, even weak preferential attachment introduces a power-law tail to the degree distribution. Additionally, this generalisation retains a great deal of the tractability of the original along with a surprising range of behaviour, although key mathematical features are modified for finite m. In particular, the critical edge density, δc which marks the onset of a giant network component is reduced with increasing tendency for preferential attachment. The positive degree-degree correlation introduced by the unbiased growth process is offset by the skewed degree distribution, reducing the network assortativity.

  18. The Probabilistic Nature of Preferential Choice

    ERIC Educational Resources Information Center

    Rieskamp, Jorg

    2008-01-01

    Previous research has developed a variety of theories explaining when and why people's decisions under risk deviate from the standard economic view of expected utility maximization. These theories are limited in their predictive accuracy in that they do not explain the probabilistic nature of preferential choice, that is, why an individual makes…

  19. 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

  20. Dogs recognize dog and human emotions.

    PubMed

    Albuquerque, Natalia; Guo, Kun; Wilkinson, Anna; Savalli, Carine; Otta, Emma; Mills, Daniel

    2016-01-01

    The perception of emotional expressions allows animals to evaluate the social intentions and motivations of each other. This usually takes place within species; however, in the case of domestic dogs, it might be advantageous to recognize the emotions of humans as well as other dogs. In this sense, the combination of visual and auditory cues to categorize others' emotions facilitates the information processing and indicates high-level cognitive representations. Using a cross-modal preferential looking paradigm, we presented dogs with either human or dog faces with different emotional valences (happy/playful versus angry/aggressive) paired with a single vocalization from the same individual with either a positive or negative valence or Brownian noise. Dogs looked significantly longer at the face whose expression was congruent to the valence of vocalization, for both conspecifics and heterospecifics, an ability previously known only in humans. These results demonstrate that dogs can extract and integrate bimodal sensory emotional information, and discriminate between positive and negative emotions from both humans and dogs. PMID:26763220

  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. 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

  3. 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

  4. 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

  5. Vaccinia virus-specific CD8+ T cell responses target a group of epitopes without a strong immunodominance hierarchy in humans

    PubMed Central

    Terajima, Masanori; Orphin, Laura; Leporati, Anita M.; Pazoles, Pamela; Cruz, John; Rothman, Alan L.; Ennis, Francis A.

    2008-01-01

    Immunization with vaccinia virus (VACV) resulted in long-lasting protection against smallpox and successful global eradication of the disease. VACV elicits strong cellular as well as humoral immune responses. Although neutralizing antibody is essential for protection, cellular immunity seems to be more important for recovery from infection in humans. We analyzed the immunodominance hierarchy of 73 previously identified VACV human CD8+ T cell epitopes restricted by HLA-A1, A2, A3, A24, B7 or B44 alleles or the alleles belonging to one of these supertypes in 56 donors after primary VACV immunization. Except for the responses to HLA-A24 supertype-restricted epitopes, there were no consistent patterns of epitope immunodominance among donors sharing the same HLA alleles or supertypes, which is in sharp contrast with the mouse studies. We, however, identified 12 epitopes that were recognized by ≥20% of donors sharing the same HLA allele; six of these contributed ≥20% of the total VACV-specific T cell response in at least one individual. VACV-specific CD8+ T cell responses targeted a group of epitopes, “relatively dominant” epitopes, without a strong immunodominance hierarchy in humans, which may be advantageous to humans to prevent the emergence of T cell escape mutants. PMID:18955096

  6. 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

  7. 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

  8. 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.

  9. Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutants.

    PubMed Central

    Okahashi, N; Takahashi, I; Nakai, M; Senpuku, H; Nisizawa, T; Koga, T

    1993-01-01

    A surface protein antigen (PAc) of Streptococcus mutans with a molecular mass of 190 kDa is considered to play an important role in the initial attachment of this streptococcus to the tooth surface. Two internal repeating amino acid sequences are present in the PAc molecule. One repeating region located in the N-terminal region is rich in alanine (A-region), and the other, located in the central region, is rich in proline (P-region). To identify antigenic epitopes on the A-region of the PAc protein, 82 sequential overlapping synthetic decapeptides covering one of the repetitive units of the A-region were synthesized. In the epitope scanning analyses using murine antisera raised against recombinant PAc (rPAc), multiple antigenic epitopes were found in the repetitive unit of the A-region, and some of them reacted with antisera to rPAc from BALB/c, B10, B10.D2, and B10.BR mice. In particular, a peptide YEAALKQY (residues 366 to 373) was recognized by anti-rPAc sera from all four strains of mice. The reactivities of anti-rPAc sera in the epitope scanning were confirmed by using a purified synthetic peptide, NAKATYEAALKQYEADLAA (corresponding to residues 361 to 379). Furthermore, antisera against a surface protein antigen PAg (SpaA) of Streptococcus sobrinus from BALB/c mice reacted strongly to residues 330 to 337, 362 to 369, and 366 to 373 of the PAc protein by the epitope scanning analysis. An AKATYEAALKQY (residues 362 to 373 of the PAc protein)-like sequence, AKANYEAKLAQY, was found within the A-region of S. sobrinus PAg, suggesting that the amino acid sequences AKA-YEA and YEA-L-QY may be major cross-reactive epitopes of the S. mutans PAc protein and the S. sobrinus PAg protein. PMID:7681043

  10. Recognizing Prefixes in Scientific Quantities

    ERIC Educational Resources Information Center

    Sokolowski, Andrzej

    2015-01-01

    Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to…

  11. Do You Recognize This Parent?

    ERIC Educational Resources Information Center

    Wallace, Edna

    1997-01-01

    Suggests effective ways to work with parents who may be permissive, busy, detached, overprotective, or negative. Recommends that child care professionals be sensitive and understanding, recognize other demands on parents' time and communicate competitively with them, use terms parents understand, accept various levels of parental involvement, be…

  12. Recognizing and Managing Interpersonal Conflict.

    ERIC Educational Resources Information Center

    Deane, Nancy; Hovland, Michael

    1993-01-01

    Practical advice is offered, to managers and supervisors at any level, on recognizing and analyzing interpersonal conflicts, managing such conflicts and making them productive, and ensuring that performance reviews result in progress for both supervisor and employee. Conflict is seen as inevitable, an opportunity to take action, and manageable.…

  13. 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

  14. Photonic devices based on preferential etching.

    PubMed

    Bellini, Bob; Larchanché, Jean-François; Vilcot, Jean-Pierre; Decoster, Didier; Beccherelli, Romeo; d'Alessandro, Antonio

    2005-11-20

    We introduce a design concept of optical waveguides characterized by a practical and reproducible process based on preferential etching of crystalline silicon substrates. Low-loss waveguides, spot-size converters, and power dividers have been obtained with polymers. We have also aligned liquid crystals in the waveguides and demonstrated guided propagation. Therefore this technology is a suitable platform for soft-matter photonics and heterogeneous integration. PMID:16318190

  15. Discovering Preferential Patterns in Sectoral Trade Networks

    PubMed Central

    Cingolani, Isabella; Piccardi, Carlo; Tajoli, Lucia

    2015-01-01

    We analyze the patterns of import/export bilateral relations, with the aim of assessing the relevance and shape of “preferentiality” in countries’ trade decisions. Preferentiality here is defined as the tendency to concentrate trade on one or few partners. With this purpose, we adopt a systemic approach through the use of the tools of complex network analysis. In particular, we apply a pattern detection approach based on community and pseudocommunity analysis, in order to highlight the groups of countries within which most of members’ trade occur. The method is applied to two intra-industry trade networks consisting of 221 countries, relative to the low-tech “Textiles and Textile Articles” and the high-tech “Electronics” sectors for the year 2006, to look at the structure of world trade before the start of the international financial crisis. It turns out that the two networks display some similarities and some differences in preferential trade patterns: they both include few significant communities that define narrow sets of countries trading with each other as preferential destinations markets or supply sources, and they are characterized by the presence of similar hierarchical structures, led by the largest economies. But there are also distinctive features due to the characteristics of the industries examined, in which the organization of production and the destination markets are different. Overall, the extent of preferentiality and partner selection at the sector level confirm the relevance of international trade costs still today, inducing countries to seek the highest efficiency in their trade patterns. PMID:26485163

  16. Human CD8(+) T Cells Target Multiple Epitopes in Respiratory Syncytial Virus Polymerase.

    PubMed

    Burbulla, Daniel; Günther, Patrick S; Peper, Janet K; Jahn, Gerhard; Dennehy, Kevin M

    2016-06-01

    Respiratory syncytial virus (RSV) infection is a serious health problem in young children, immunocompromised patients, and the elderly. The development of novel prevention strategies, such as a vaccine to RSV, is a high priority. One strategy is to design a peptide-based vaccine that activates appropriate CD8(+) T-cell responses. However, this approach is limited by the low number of RSV peptide epitopes defined to date that activate CD8(+) T cells. We aimed to identify peptide epitopes that are presented by common human leukocyte antigen types (HLA-A*01, -A*02, and -B*07). We identify one novel HLA-A*02-restricted and two novel HLA-A*01-restricted peptide epitopes from RSV polymerase. Peptide-HLA multimer staining of specific T cells from healthy donor peripheral blood mononuclear cell, the memory phenotype of such peptide-specific T cells ex vivo, and functional IFNγ responses in short-term stimulation assays suggest that these peptides are recognized during RSV infection. Such peptides are candidates for inclusion into a peptide-based RSV vaccine designed to stimulate defined CD8(+) T-cell responses. PMID:27070377

  17. [Preparation of epitope imprinted particles for transferrin recognition by reversible addition fragmentation chain transfer strategy].

    PubMed

    Li, Qinran; Yang, Kaiguang; Li, Senwu; Liu, Jianxi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-10-01

    A kind of novel epitope surface imprinted particles was prepared by the reversible addition fragmentation chain transfer (RAFT) strategy. The epitope of transferrin, N-terminal peptide of the protein with nine amino acid residues, was chosen as the template and immobi- lized with covalent interaction on the surface of silica particles through the truss arm glutaraldehyde. The living/controlled polymerization was initialed by 2,2'-azobisisobutyronitrile (AIBN) at 70 °C in the solution of N,N-dimethylformamide, with the regulation by triothioester agent 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid. Methacrylic acid and 2-hydroxyethyl methacrylate were chosen as the functional monomers and N, N-methylenebisacrylamide was chosen as the cross-linker in this polymerization. For this material, the binding capacity of the nine residue peptide could reach 2.36 mg/g with the imprinting factor (IF) of 1.89, while that for transferrin could reach 4.98 mg/g with IF of 1.61. The equilibrium could be achieved in 120 min for the transferrin recognition. In multi-protein competitive recognition, the imprinted factor of transferrin was the highest in the mixture of transferrin and other competitive proteins, such as cytochrome C and β-lactoglobulin. The results indicated that these epitope surface imprinted particles with RAFT strategy could recognize not only the nine residue peptide but also the transferrin with good selectivity, high binding capacity and fast mass transfer. PMID:25739262

  18. Oxidation-specific epitopes as targets for biotheranostic applications in humans: Biomarkers, molecular imaging and therapeutics

    PubMed Central

    Miller, Yury I.; Tsimikas, Sotirios

    2014-01-01

    Purpose of Review Emerging data demonstrates the potential of translational applications of antibodies directed against oxidation-specific epitopes (OSE). “Biotheranostics” in cardiovascular disease (CVD) describes targeting of OSE for biomarker, therapeutic and molecular imaging diagnostic applications. Recent findings Lipid oxidation collectively yields a large variety of oxidation-specific epitopes (OSE), such as oxidized phospholipids (OxPL) and malondialdehyde (MDA) epitopes. OSE are immunogenic, pro-inflammatory, pro-atherogenic and plaque destabilizing and represent danger associated molecular patterns (DAMPs). DAMPs are recognized by the innate immune system via pattern recognition receptors, including scavenger receptors IgM natural antibodies and complement factor H (CFH), that bind, neutralize and/or facilitate their clearance. Biomarker assays measuring OxPL present on apolipoprotein B-100 lipoproteins, and particularly on lipoprotein (a), predict the development of CVD events. In contrast, OxPL on plasminogen facilitate fibrinolysis and may reduce atherothrombosis. Oxidation-specific antibodies (OSA) attached to magnetic nanoparticles image lipid-rich, oxidation-rich plaques. Infusion or overexpression of OSA reduces the progression of atherosclerosis, suggesting that they may be used in similar applications in humans. Summary Using the accelerating knowledge base and improved understanding of the interplay of oxidation, inflammation and innate and adaptive immunity in atherogenesis, emerging clinical applications of OSA may identify, monitor and treat CVD in humans. PMID:23995232

  19. 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

  20. 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

  1. Fusion of Pedigreed Preferential Relations as Beliefs

    NASA Astrophysics Data System (ADS)

    Suzuki, Yoshitaka; Tojo, Satoshi

    Belief fusion, instead of AGM belief revision, was first proposed to solve the problem of inconsistency, that arised from repetitive application of the operation when agents' knowledge were amalgamated. In the preceding work of Maynard-Reid II and Shoham, the fusion operator is applied to belief states, which is total preorders over possible worlds which is based on the semantics of belief revision. Moreover, they introduced the pedigreed belief state, which represented multiple sources of belief states, ordered by a credibility ranking. However in the theory, all the sources must be totally ordered and thus applicable area is quite restrictive. In this paper, we realize the fusion operator of multiple agents for partially ordered sources. When we consider such a partial ranking over sources, there is no need to restrict that each agent has total preorders over possible worlds. The preferential model, based on the semantics on nonmonotonic reasoning, allows each agent to have strict partial orders over possible worlds. Especially, such an order is called a preferential relation, that prescribes a world is more plausible than the other. Therefore, we introduce an operation which combines multiple preferential relations of agents. In addition, we show that our operation can properly include the ordinary belief fusion.

  2. Do Infants Recognize the Arcimboldo Images as Faces? Behavioral and Near-Infrared Spectroscopic Study

    ERIC Educational Resources Information Center

    Kobayashi, Megumi; Otsuka, Yumiko; Nakato, Emi; Kanazawa, So; Yamaguchi, Masami K.; Kakigi, Ryusuke

    2012-01-01

    Arcimboldo images induce the perception of faces when shown upright despite the fact that only nonfacial objects such as vegetables and fruits are painted. In the current study, we examined whether infants recognize a face in the Arcimboldo images by using the preferential looking technique and near-infrared spectroscopy (NIRS). In the first…

  3. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    PubMed Central

    Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

    2009-01-01

    Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the

  4. 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

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

    PubMed Central

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

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  6. Expression of Epitope-Tagged Proteins in Mammalian Cells in Culture.

    PubMed

    Bhatt, Jay M; Styers, Melanie L; Sztul, Elizabeth

    2016-01-01

    Before the advent of molecular methods to tag proteins, visualization of proteins within cells required the use of antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Epitope tagging allows the detection of all proteins with existing sequence information, irrespective of the availability of antibodies directed against them. This technique involves the generation of DNA constructs that express the protein of interest tagged with an epitope that can be recognized by a commercially available antibody. Proteins can be tagged with a wide variety of epitopes using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into mammalian cell lines and, in most cases, tightly mimic the behavior of the endogenous protein. Tagged proteins exogenously expressed in cells provide different types of information depending on the subsequent detection approaches. Using immunofluorescence and immunoelectron microscopy with anti-tag antibodies, relative to known markers of cellular organelles, can provide information on the subcellular localization of the tagged protein and may provide clues regarding the protein's function. Immunofluorescence with anti-tag antibodies can also be utilized to assess the tagged protein's responses to cellular signals and pharmacological treatments. Immunoprecipitations with anti-tag antibodies can recover protein complexes containing the protein of interest, resulting in the identification of interacting proteins. Recovery of tagged proteins on affinity matrices allows their purification for use in biochemical assays. In addition, specialized fluorescent tags, such as the green fluorescent protein (GFP) allow the analysis of cellular dynamics in live cells in real time. PMID:27515071

  7. Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alzheimer neurofibrillary tangles.

    PubMed Central

    Ksiezak-Reding, H; Dickson, D W; Davies, P; Yen, S H

    1987-01-01

    Eleven anti-neurofilament (anti-NF) monoclonal antibodies were studied for their reactivity with heat-stable, microtubule-associated proteins and Alzheimer neurofibrillary tangles (ANT). On immunoblots of NF proteins, the antibodies recognized epitopes that were variably sensitive to Escherichia coli alkaline phosphatase. Eight of the antibodies showed reactivity with ANT and decreased binding to electroblotted NF after phosphatase treatment. The same eight antibodies reacted with tau proteins from bovine and rat brain, binding to tau proteins was also substantially reduced by phosphatase. Of the eight antibodies that bound to animal tau proteins, five also bound to tau proteins from normal human brain. All of the antibodies that bound to animal tau proteins stained ANT in frozen tissue sections. Brief treatment of tissue sections with trypsin in most cases enhanced antibody binding to ANT. All antibodies that lacked reactivity with tau proteins failed to bind ANT. Phosphatase treatment of Alzheimer tissue sections did not change the immunoreactivity of ANT and neurites in senile plaques with ANT-reactive, anti-NF antibodies, except for two antibodies that showed decreased binding to ANT. In contrast, axonal staining was decreased or eliminated by phosphatase treatment, similar to the response of electroblotted NF and tau proteins. These results suggest that staining of ANT by anti-NF antibodies may be due to cross-reaction of anti-NF with epitopes in tau proteins, the epitopes in axons, NF, and tau are sensitive to the effect of phosphatase, whereas the majority of those in ANT are not, and some of the epitopes in ANT that are shared with NF and tau proteins are not readily accessible to antibody binding. Images PMID:2437579

  8. 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

  9. 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

  10. Recognizing Body Dysmorphic Disorder (Dysmorphophobia)

    PubMed Central

    Varma, Anukriti; Rastogi, Rajesh

    2015-01-01

    Dysmorphophobia is a psychiatric condition which frequently presents in the clinics of dermatologists and plastic surgeons. This disorder (also called body dysmorphic disorder) is troublesome to the patient whilst being confusing for the doctor. This commonly undiagnosed condition can be detected by a few simple steps. Timely referral to a psychiatrist benefits most patients suffering from it. This article describes with a case vignette, how to recognize body dysmorphic disorder presenting in the dermatological or aesthetic surgery set up. Diagnostic criteria, eitiology, approach to patient, management strategy and when to refer are important learning points. The importance of recognizing this disorder timely and referring the patient to the psychiatrist for appropriate treatment is crucial. This article covers all aspects of body dysmorphic disorder relevant to dermatologists and plastic surgeons and hopes to be useful in a better understanding of this disorder. PMID:26644741

  11. Recognizing Prefixes in Scientific Quantities

    NASA Astrophysics Data System (ADS)

    Sokolowski, Andrzej

    2015-09-01

    Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to meters." Similar students' mistakes were reported also by AP Chemistry readers "as in previous years, students still had difficulty converting kJ to J." While traditional teaching focuses on memorizing the symbols of prefixes, little attention is given to helping learners recognize a prefix in a given quantity. I noticed in my teaching practice that by making the processes of identifying prefixes more explicit, students make fewer mistakes on unit conversion. Thus, this paper presents an outline of a lesson that focuses on prefix recognition. It is designed for a first-year college physics class; however, its key points can be addressed to any group of physics students.

  12. Recognizing Body Dysmorphic Disorder (Dysmorphophobia).

    PubMed

    Varma, Anukriti; Rastogi, Rajesh

    2015-01-01

    Dysmorphophobia is a psychiatric condition which frequently presents in the clinics of dermatologists and plastic surgeons. This disorder (also called body dysmorphic disorder) is troublesome to the patient whilst being confusing for the doctor. This commonly undiagnosed condition can be detected by a few simple steps. Timely referral to a psychiatrist benefits most patients suffering from it. This article describes with a case vignette, how to recognize body dysmorphic disorder presenting in the dermatological or aesthetic surgery set up. Diagnostic criteria, eitiology, approach to patient, management strategy and when to refer are important learning points. The importance of recognizing this disorder timely and referring the patient to the psychiatrist for appropriate treatment is crucial. This article covers all aspects of body dysmorphic disorder relevant to dermatologists and plastic surgeons and hopes to be useful in a better understanding of this disorder. PMID:26644741

  13. Plant-based Production of Two Chimeric Monoclonal IgG Antibodies Directed against Immunodominant Epitopes of Vibrio cholerae Lipopolysaccharide

    PubMed Central

    Levinson, Kara J.; Giffen, Samantha R.; Pauly, Michael H.; Kim, Do H.; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J.; Zeitlin, Larry; Mantis, Nicholas J.

    2015-01-01

    We have produced and characterized two chimeric IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest. PMID:25865265

  14. Therapeutic Vaccination against the Rhesus Lymphocryptovirus EBNA-1 Homologue, rhEBNA-1, Elicits T Cell Responses to Novel Epitopes in Rhesus Macaques

    PubMed Central

    Silveira, Eduardo L. V.; Fogg, Mark H.; Leskowitz, Rachel M.; Ertl, Hildegund C.; Wiseman, Roger W.; O'Connor, David H.; Lieberman, Paul; Wang, Fred

    2013-01-01

    Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC–rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination. PMID:24089556

  15. Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

    PubMed Central

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease. PMID:24015196

  16. A case study of preferential bestiality (zoophilia).

    PubMed

    Earls, Christopher M; Lalumière, Martin L

    2002-01-01

    Humans show a wide array of sexual preferences and behaviors. Although most humans prefer and have sex with consenting adults of the opposite sex, some individuals have unconventional preferences with regard to the sex or age of sexual partners, or with regard to the nature of sexual activities. In this paper, we describe a rare case of preferential bestiality, or zoophilia. The client meets the most stringent criteria for the diagnosis of zoophilia. In particular, his phallometrically measured arousal pattern shows a sexual preference for horses over other species, including humans. PMID:11803597

  17. Emergence of social structures via preferential selection

    NASA Astrophysics Data System (ADS)

    Lipowski, Adam; Lipowska, Dorota; Ferreira, Antonio Luis

    2014-09-01

    We examine a weighted-network multiagent model with preferential selection such that agents choose partners with probability p (w), where w is the number of their past selections. When p (w) increases sublinearly with the number of past selections [p(w)˜wα,α<1], agents develop a uniform preference for all other agents. At α =1, this state loses stability and more complex structures form. For a superlinear increase (α>1), strong heterogeneities emerge and agents make selections mainly within small and sometimes asymmetric clusters. Even in a few-agent case, the formation of such clusters resembles phase transitions with spontaneous symmetry breaking.

  18. Ultrastructural localization of beta-amyloid, tau, and ubiquitin epitopes in extracellular neurofibrillary tangles.

    PubMed Central

    Tabaton, M; Cammarata, S; Mancardi, G; Manetto, V; Autilio-Gambetti, L; Perry, G; Gambetti, P

    1991-01-01

    Neurofibrillary tangles (NFTs), a hallmark of Alzheimer disease, are commonly located in perikarya of neurons. In advanced cases of Alzheimer disease, however, NFTs are observed also in the extracellular space. As extracellular NFTs (E-NFTs), and occasionally intracellular NFTs (I-NFTs), are recognized by antibodies to beta-amyloid protein (beta AP), beta AP may be present not only in amyloid deposits but also in paired helical filaments (PHFs), the primary components of NFTs. We compared the antigenic characteristics of I-NFTs and E-NFTs with light- and electron-microscopic immunocytochemistry by using several antibodies to noncontiguous epitopes of the microtubule-associated protein tau and of ubiquitin (Ub) as well as an antiserum to beta AP. At variance with I-NFTs, E-NFTs were made predominantly of straight filaments (SFs), rather than PHFs, that were often separated by astroglial processes and in close association with small beta AP deposits. Occasionally, E-NFTs were made of bundles of amorphous material, which showed no resemblance to SFs, PHFs, or amyloid fibrils. The antigenic changes in E-NFTs suggest that when NFTs become extracellular they lose the N and, possibly, the C termini of tau while maintaining the intermediate region of the molecule; they also lose the N-terminal two-thirds of Ub while the C-terminal conjugation site of Ub is preserved. A small subset of E-NFTs reacted with antibodies to both beta AP and tau. Although in most E-NFTs, the epitopes recognized by tau and Ub antibodies were located in typical PHFs and SFs, the epitopes recognized in this subset of anti-beta AP and anti-tau-positive E-NFTs were located exclusively in the bundles of amorphous material. It is suggested that either beta AP epitopes are present but inaccessible in PHFs and SFs and become exposed after conformational changes occurring in the extracellular space or PHFs and SFs become closely associated with beta AP in the extracellular space. Images PMID:1706517

  19. Mutant MHC class II epitopes drive therapeutic immune responses to cancer

    PubMed Central

    Kreiter, Sebastian; Vormehr, Mathias; van de Roemer, Niels; Diken, Mustafa; Löwer, Martin; Diekmann, Jan; Boegel, Sebastian; Schrörs, Barbara; Vascotto, Fulvia; Castle, John C.; Tadmor, Arbel D.; Schoenberger, Stephen P.; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope

  20. Preventing and Recognizing Prescription Drug Abuse

    MedlinePlus

    ... Abuse » Preventing and recognizing prescription drug abuse Prescription Drug Abuse Email Facebook Twitter Preventing and recognizing prescription drug abuse To ensure proper medical care, patients should discuss ...

  1. Brandon RHA recognized for energy efficiency.

    PubMed

    Waddington, Kent; Neal, Gordon

    2002-01-01

    In a recent national competition recognizing leadership in energy efficiency and greenhouse gas education, Brandon Regional Health Authority was recognized for conscientious use of resources. PMID:12357581

  2. Recognizing Patterns in Debris Disks

    NASA Technical Reports Server (NTRS)

    Kuchner, Marc

    2009-01-01

    An extrasolar planet sculpts the famous debris dish around Fomalhaut; probably many other debris disks contain planets that we could locate if only we could better recognize their signatures in the dust that surrounds them. I will describe the latest 3-D models of debris dish dynamics / models that include planets, grain-grain collisions and even ISM-disk interactions. I will show why all these ingredients are needed to explain disk images--and what the images are telling us about planet formation.

  3. Preferential Flow in Fractured Welded Tuffs

    SciTech Connect

    Salve, Rohit

    2004-08-15

    To better understand preferential flow in fractured rock, we carried out an in situ field experiment in the Exploratory Studies Facility at Yucca Mountain, Nevada. This experiment involved the release of {approx} 22 m3 of ponded water (at a pressure head of {approx} 0.04 m) over a period of 7 months, directly onto a 12 m2 infiltration plot located on a fractured welded tuff surface. As water was released, changes in moisture content were monitored along horizontal boreholes located in the formation {approx} 19-22 m below. Distinct flow zones, varying in flow velocity, wetted cross-sectional area, and extent of lateral movement, intercepted the monitoring boreholes. There was also evidence of water being diverted above the ceiling of a cavity in the immediate vicinity of the monitoring boreholes. Observations from this field experiment suggest that isolated conduits, each encompassing a large number of fractures, develop within the fractured rock formation to form preferential flow paths that persist if there is a continuous supply of water. In addition, in fractured welded tuffs the propensity for fracture-matrix interactions is significantly greater than that suggested by existing conceptual models,in which flow occurs along a section of fracture surfaces. An overriding conclusion is that field investigations at spatial scales of tens of meters provide data critical to the fundamental understanding of flow in fractured rock.

  4. Accelerating networks: Effects of preferential connections

    NASA Astrophysics Data System (ADS)

    Jeon, Y.-P.; McCoy, B. J.

    2007-12-01

    Networks are commonly observed structures in complex systems with interacting and interdependent parts that self-organize. For nonlinearly growing networks, when the total number of connections increases faster than the total number of nodes, the network is said to accelerate. We propose a systematic model for the dynamics of growing networks represented by distribution kinetics equations. We define the nodal-linkage distribution, construct a population dynamics equation based on the association-dissociation process, and perform the moment calculations to describe the dynamics of such networks. For nondirectional networks with finite numbers of nodes and connections, the moments are the total number of nodes, the total number of connections, and the degree (the average number of connections per node), represented by the average moment. Size independent rate coefficients yield an exponential network describing the network without preferential attachment, and size dependent rate coefficients produce a power law network with preferential attachment. The model quantitatively describes accelerating network growth data for a supercomputer (Earth Simulator), for regulatory gene networks, and for the Internet.

  5. 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.

  6. Predicting Interaction Sites from the Energetics of Isolated Proteins: A New Approach to Epitope Mapping

    PubMed Central

    Scarabelli, Guido; Morra, Giulia; Colombo, Giorgio

    2010-01-01

    Abstract An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design. PMID:20441761

  7. IMMUNODOMINANT EPITOPE AND PROPERTIES OF PYROGLUTAMATE-MODIFIED Aβ-SPECIFIC ANTIBODIES PRODUCED IN RABBITS

    PubMed Central

    Acero, G.; Manoutcharian, K.; Vasilevko, V.; Munguia, M.E.; Govezensky, T.; Coronas, G.; Luz-Madrigal, A.; Cribbs, DH.; Gevorkian, G.

    2009-01-01

    N-truncated and N-modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer’s disease (AD) and Down’s syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Aβ is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full-length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 3 (AβN3(pE)). We demonstrated that AβN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AβN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AβN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AβN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:19545911

  8. Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits.

    PubMed

    Acero, G; Manoutcharian, K; Vasilevko, V; Munguia, M E; Govezensky, T; Coronas, G; Luz-Madrigal, A; Cribbs, D H; Gevorkian, G

    2009-08-18

    N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides. PMID:19545911

  9. 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

  10. 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

  11. Antarctic skuas recognize individual humans.

    PubMed

    Lee, Won Young; Han, Yeong-Deok; Lee, Sang-Im; Jablonski, Piotr G; Jung, Jin-Woo; Kim, Jeong-Hoon

    2016-07-01

    Recent findings report that wild animals can recognize individual humans. To explain how the animals distinguish humans, two hypotheses are proposed. The high cognitive abilities hypothesis implies that pre-existing high intelligence enabled animals to acquire such abilities. The pre-exposure to stimuli hypothesis suggests that frequent encounters with humans promote the acquisition of discriminatory abilities in these species. Here, we examine individual human recognition abilities in a wild Antarctic species, the brown skua (Stercorarius antarcticus), which lives away from typical human settlements and was only recently exposed to humans due to activities at Antarctic stations. We found that, as nest visits were repeated, the skua parents responded at further distances and were more likely to attack the nest intruder. Also, we demonstrated that seven out of seven breeding pairs of skuas selectively responded to a human nest intruder with aggression and ignored a neutral human who had not previously approached the nest. The results indicate that Antarctic skuas, a species that typically inhabited in human-free areas, are able to recognize individual humans who disturbed their nests. Our findings generally support the high cognitive abilities hypothesis, but this ability can be acquired during a relatively short period in the life of an individual as a result of interactions between individual birds and humans. PMID:26939544

  12. Recognizing new medical knowledge computationally.

    PubMed Central

    Nelson, S. J.; Cole, W. G.; Tuttle, M. S.; Olson, N. E.; Sherertz, D. D.

    1993-01-01

    Can new medical knowledge be recognized computationally? We know knowledge is changing, and our knowledge-based systems will need to accommodate that change in knowledge on a regular basis if they are to stay successful. Computational recognition of these changes seems desirable. It is unlikely that low level objects in the computational universe, bits and characters, will change much over time, higher level objects of language, where meaning begins to emerge, may show change. An analysis of ten arbitrarily selected paragraphs from the Medical Knowledge Self-Assessment Program of the American College of Physicians was used as a test bed for nominal phrase recognition. While there were words not known to Meta-1.2, only 8 of the 32 concepts new to the primary author were pointed to by new words. Use of a barrier word method was successful in identifying 23 of the 32 new concepts. Use of co-occurrence (in sentences) of putative nominal phrases may reduce the amount of human effort involved in recognizing the emergence of new relationships. PMID:8130505

  13. Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain

    PubMed Central

    Mao, Xiaoyan; Zhang, Tiancheng; Ji, Guanghui; Shi, Xin; Xia, Tian; Lu, Weijia; Zhang, Dapeng; Dai, Jianxin; Guo, Yajun

    2012-01-01

    Background Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. Methods and Findings We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. Conclusions The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes. PMID:22952786

  14. The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: a carbohydrate of unique evolution and clinical relevance

    PubMed Central

    Macher, Bruce A.; Galili, Uri

    2008-01-01

    In 1985, we reported that a naturally occurring human antibody (anti-Gal), produced as the most abundant antibody (1% of immunoglobulins) throughout the life of all individuals, recognizes a carbohydrate epitope Galα1-3Galβ1-4GlcNAc-R (the α-gal epitope). Since that time, an extensive literature has developed on discoveries related to the α-gal epitope and the anti-Gal antibody, including the barrier they form in xenotransplantation and their reciprocity in mammalian evolution. This review covers these topics and new avenues of clinical importance related to this (α-gal epitope/anti-Gal) unique antigen/antibody system in improving the efficacy of viral vaccines and in immunotherapy against cancer. PMID:18047841

  15. 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

  16. Preferential brain homing following intranasal administration of Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey; Pereiraperrin, Mercio

    2009-04-01

    The Chagas' disease parasite Trypanosoma cruzi commonly infects humans through skin abrasions or mucosa from reduviid bug excreta. Yet most studies on animal models start with subcutaneous or intraperitoneal injections, a distant approximation of the skin abrasion route. We show here that atraumatic placement of T. cruzi in the mouse nasal cavity produced low parasitemia, high survival rates, and preferential brain invasion compared to the case with subcutaneously injected parasites. Brain invasion was particularly prominent in the basal ganglia, peaked at a time when parasitemia was no longer detectable, and elicited a relatively large number of inflammatory foci. Yet, based on motor behavioral parameters and staining with Fluoro-Jade C, a dye that specifically recognizes apoptotic and necrotic neurons, brain invasion did not cause neurodegenerative events, in contrast to the neurodegeneration in the enteric nervous system. The results indicate that placement of T. cruzi on the mucosa in the mouse nasal cavity establishes a systemic infection with a robust yet harmless infection of the brain, seemingly analogous to disease progression in humans. The model may facilitate studies designed to understand mechanisms underlying T. cruzi infection of the central nervous system. PMID:19168740

  17. Preferential Brain Homing following Intranasal Administration of Trypanosoma cruzi▿

    PubMed Central

    Caradonna, Kacey; PereiraPerrin, Mercio

    2009-01-01

    The Chagas’ disease parasite Trypanosoma cruzi commonly infects humans through skin abrasions or mucosa from reduviid bug excreta. Yet most studies on animal models start with subcutaneous or intraperitoneal injections, a distant approximation of the skin abrasion route. We show here that atraumatic placement of T. cruzi in the mouse nasal cavity produced low parasitemia, high survival rates, and preferential brain invasion compared to the case with subcutaneously injected parasites. Brain invasion was particularly prominent in the basal ganglia, peaked at a time when parasitemia was no longer detectable, and elicited a relatively large number of inflammatory foci. Yet, based on motor behavioral parameters and staining with Fluoro-Jade C, a dye that specifically recognizes apoptotic and necrotic neurons, brain invasion did not cause neurodegenerative events, in contrast to the neurodegeneration in the enteric nervous system. The results indicate that placement of T. cruzi on the mucosa in the mouse nasal cavity establishes a systemic infection with a robust yet harmless infection of the brain, seemingly analogous to disease progression in humans. The model may facilitate studies designed to understand mechanisms underlying T. cruzi infection of the central nervous system. PMID:19168740

  18. 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

  19. 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

  20. 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

  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. A case study of preferential bestiality.

    PubMed

    Earls, Christopher M; Lalumière, Martin L

    2009-08-01

    In a previous article, we presented phallometric data to illustrate a case of preferential bestiality or zoophilia (Earls & Lalumière, Sex Abuse: J Res Treat, 14:83-88, 2002). Based on the available literature, we argued that a marked preference for having sex with animals over sex with humans is extremely rare. In the present article, we describe a second case of zoophilia that challenges the widely held assumptions that men who have sex with animals are generally of below average intelligence and come from rural areas. In addition, we provide a brief review of a burgeoning quantitative literature using large groups of zoophiles recruited from internet sources. Although estimates of the prevalence of zoophilia are not possible at this time, it appears that zoophilia is not as rare as once thought and shares many features with other atypical sexual interests. PMID:18157625

  3. Growth fluctuation in preferential attachment dynamics

    NASA Astrophysics Data System (ADS)

    Hashimoto, Yasuhiro

    2016-04-01

    In the Yule-Simon process, creation and selection of words follows the preferential attachment mechanism, resulting in a power-law growth in the cumulative number of individual word occurrences as well as the power-law population distribution of the vocabulary. This is derived using mean-field approximation, assuming a continuum limit of both the time and number of word occurrences. However, time and word occurrences are inherently discrete in the process, and it is natural to assume that the cumulative number of word occurrences has a certain fluctuation around the average behavior predicted by the mean-field approximation. We derive the exact and approximate forms of the probability distribution of such fluctuation analytically, and confirm that those probability distributions are well supported by the numerical experiments.

  4. Preferential attachment in multiple trade networks

    NASA Astrophysics Data System (ADS)

    Foschi, Rachele; Riccaboni, Massimo; Schiavo, Stefano

    2014-08-01

    In this paper we develop a model for the evolution of multiple networks which is able to replicate the concentrated and sparse nature of world trade data. Our model is an extension of the preferential attachment growth model to the case of multiple networks. Countries trade a variety of goods of different complexity. Every country progressively evolves from trading less sophisticated to high-tech goods. The probabilities of capturing more trade opportunities at a given level of complexity and of starting to trade more complex goods are both proportional to the number of existing trade links. We provide a set of theoretical predictions and simulative results. A calibration exercise shows that our model replicates the same concentration level of world trade as well as the sparsity pattern of the trade matrix. We also discuss a set of numerical solutions to deal with large multiple networks.

  5. 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

  6. 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

  7. Efficient Generation of a Hepatitis B Virus Cytotoxic T Lymphocyte Epitope Requires the Structural Features of Immunoproteasomes

    PubMed Central

    Sijts, Alice J.A.M.; Ruppert, Thomas; Rehermann, Barbara; Schmidt, Marion; Koszinowski, Ulrich; Kloetzel, Peter-M.

    2000-01-01

    Interferon (IFN)-γ–induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex–like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68–restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-γ–induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity. PMID:10662796

  8. Heligmosomoides polygyrus elicits a dominant non-protective antibody response directed against restricted glycan and peptide epitopes

    PubMed Central

    Hewitson, James P; Filbey, Kara J; Grainger, John R; Dowle, Adam A; Pearson, Mark; Murray, Janice; Harcus, Yvonne; Maizels, Rick M

    2014-01-01

    Summary Heligmosomoides polygyrus is a widely-used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary antibody response as a subset of the secreted products in H. polygyrus Excretory-Secretory (HES) antigen. An immunodominant epitope is an O-linked glycan (named Glycan A) carried on 3 highly-expressed HES glycoproteins (VAL-1, -2 and -5), which stimulates only IgM antibodies, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (Glycan B), present on both a non-protein high molecular weight component (HM) and a 65-kDa molecule, is widely distributed in adult somatic tissues; while HM-65 molecules bear phosphorylcholine (PC), the Glycan B epitope itself is not PC. Class-switched IgG1 antibodies are found to Glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary antibody responses are similar while recognizing in addition VAL-7. Whilst vaccination with HES conferred complete protection against challenge H. polygyrus infection, monoclonal antibodies raised against each of the glycan epitopes, and against VAL-1, -2 and -4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective antibodies against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity. PMID:21964031

  9. Definition of a possible genetic basis for susceptibility to acute myelogenous leukemia associated with the presence of a polymorphic Ia epitope.

    PubMed Central

    Seremetis, S; Cuttner, J; Winchester, R

    1985-01-01

    The polymorphic Ia epitope recognized by monoclonal antibody 109d6 is detectable on the leukemic cells of a significantly increased number of individuals with acute myelogenous leukemia, compared with its frequency in normal healthy control individuals. In control individuals, the presence of the 109d6 epitope is closely correlated with but not identical to the DRw53 allo-specificity. However, the frequency of particular conventional Ia allodeterminants, including DRw53, is not significantly elevated in the leukemia group. Considerable evidence supports the conclusion that the high frequency of the 109d6 epitope reflects an inherited basis for susceptibility to the development of acute myelogenous leukemia and not a differentiation event occurring in the leukemic lineage. The 109d6 determinant is expressed by leukemic myeloblasts as well as by homologous normal B cells and monocytes obtained from the same individuals during remission of the leukemia. Furthermore, in healthy family members the 109d6 epitope is encoded by Ia haplotypes that are shared with the patient. Of special interest, certain of these haplotypes have combinations of the 109d6 epitope and Ia specificities not commonly seen in normal individuals; here, also, healthy family members share these haplotypes. PMID:2414320

  10. Preparation of High-Efficiency Cytochrome c-Imprinted Polymer on the Surface of Magnetic Carbon Nanotubes by Epitope Approach via Metal Chelation and Six-Membered Ring.

    PubMed

    Qin, Ya-Ping; Li, Dong-Yan; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui

    2016-04-27

    A novel epitope molecularly imprinted polymer on the surface of magnetic carbon nanotubes (MCNTs@EMIP) was successfully fabricated to specifically recognize target protein cytochrome c (Cyt C) with high performance. The peptides sequences corresponding to the surface-exposed C-terminus domains of Cyt C was selected as epitope template molecule, and commercially available zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were employed as functional monomer and cross-linker, respectively, to synthesize MIP via free radical polymerization. The epitope was immobilized via metal chelation and six-membered ring formed between the functional monomer and the hydroxyl and amino groups of the epitope. The resulting MCNTs@EMIP exhibited specific recognition ability toward target Cyt C including more satisfactory imprinting factor (about 11.7) than that of other reported imprinting methods. In addition, the MCNTs@EMIP demonstrated a high adsorption amount (about 780.0 mg g(-1)) and excellent selectivity. Besides, the magnetic property of the support material made the processes easy and highly efficient by assistance of an external magnetic field. High-performance liquid chromatography analysis of Cyt C in bovine blood real sample and protein mixture indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@EMIP had potential to be applied in bioseparation area. In brief, this study provided a new protocol to detect target protein in complex sample via epitope imprinting approach and surface imprinting strategy. PMID:27049646

  11. Recombinant infectious bursal disease virus expressing Newcastle disease virus (NDV) neutralizing epitope confers partial protection against virulent NDV challenge in chickens.

    PubMed

    Li, Kai; Gao, Li; Gao, Honglei; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2014-01-01

    In this study, the regions in the infectious bursal disease virus (IBDV) genome that are amenable to the introduction of a sequence encoding a virus-neutralizing epitope of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein were identified. By using the reverse genetics approach, insertions or substitutions of sequences encoding the NDV epitope were engineered in the exposed loops (PBC, PHI and [Formula: see text] ) of the VP2 capsid protein and the N terminus of the nonstructural VP5 protein as well as the pep7a and pep7b regions of the pVP2 precursor of a commonly used IBDV vaccine strain, Gt. Three recombinant IBDVs expressing the NDV epitopes were successfully rescued in the PBC, pep7b and VP5 regions and the expressed epitope was recognized by anti-HN antibodies. Genetic analysis showed that the IBDV recombinants carrying the NDV epitopes were stable in cell cultures and in chickens. Animal studies demonstrated that the IBDV recombinants were innocuous in chickens. Vaccination with the recombinant viruses generated antibody responses against both IBDV and NDV, and provided 70-80% protection against IBDV and 50-60% protection against NDV. These results indicate that the recombinant IBDV has the potential to serve as a novel vaccine vector for other pathogens. In future studies, it is worth considering research to improve IBDV vector vaccine to get complete protection and safety of animals and humans. PMID:24200519

  12. SplitCore Technology Allows Efficient Production of Virus-Like Particles Presenting a Receptor-Contacting Epitope of Human IgE.

    PubMed

    Baltabekova, A Zh; Shagyrova, Zh S; Kamzina, A S; Voykov, M; Zhiyenbay, Ye; Ramanculov, E M; Shustov, A V

    2015-08-01

    Immunoglobulin E (IgE) plays a central role in type I hypersensitivity including allergy and asthma. Novel treatment strategy envisages development of a therapeutic vaccine designed to elicit autologous blocking antibodies against the IgE. We sought to develop an IgE-epitope antigen that induces antibodies against a receptor-contacting epitope on human IgE molecule. We designed the VLP immunogens which utilize hepatitis B virus core protein (HBcAg) as a carrier, and present arrays of the receptor-contacting epitopes of the human IgE on their surfaces. FG loop from the IgE domain Cε3 was engineered into the HBcAg. Two constructs explore a well-established approach of insertion into a main immunodominant region of the HBcAg. Third construct is different in that the carrier is produced in a form of an assembly of two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit (SplitCore technology). No VLPs were isolated from E.coli expressing the IgE-epitope antigens with contiguous sequences. On the contrary, the SplitCore antigen carrying the FG loop efficiently formed the VLPs. Immunization of mice with the VLPs presenting receptor-contacting epitope of the IgE elicited antibodies recognizing the human IgE in ELISA. PMID:25837568

  13. CD8+ T Cells Specific for Immunodominant Trans-sialidase Epitopes Contribute to Control of Trypanosoma cruzi Infection but are Not Required for Resistance1

    PubMed Central

    Rosenberg, Charles S.; Martin, Diana L.; Tarleton, Rick L.

    2011-01-01

    CD8+ T cells are essential for controlling Trypanosoma cruzi infection. During Brazil strain infection, C57BL/6 mice expand parasite-specific CD8+ T cells recognizing the dominant TSKB20 (ANYKFTLV) and sub-dominant TSKB74 (VNYDFTLV) trans-sialidase gene (TS)-encoded epitopes with up to 40% of all CD8+ T cells specific for these epitopes. Though this is one of the largest immunodominant T cell responses described for any infection, most mice fail to clear T. cruzi and subsequently develop chronic disease. To determine if immunodominant TS-specific CD8+ T cells are necessary for resistance to infection, we epitope-tolerized mice by high-dose intravenous injections of TSKB20 or TSKB74 peptides. Tolerance induction led to deletion of TS-specific CD8+ T cells but did not prevent the expansion of other effector CD8+ T cell populations. Mice tolerized against either TSKB20 or TSKB74, or both epitopes simultaneously, exhibited transient increases in parasite loads, though ultimately they controlled the acute infection. Furthermore, BALB/c mice tolerized against the TSKD14 peptide effectively controlled acute T. cruzi infection. These data are consistent with the hypothesis that development of high frequency CD8+ T cell populations focused on TS-derived epitopes contributes to optimal control of acute infection, but is not required for the development of immune resistance. PMID:20530265

  14. 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

  15. 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

  16. 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

  17. N-Propionylated Group B Meningococcal Polysaccharide Mimics a Unique Bactericidal Capsular Epitope in Group B Neisseria meningitidis

    PubMed Central

    Pon, Robert A.; Lussier, Michele; Yang, Qing-Ling; Jennings, Harold J.

    1997-01-01

    The N-propionylated group B meningococcal polysaccharide (NPrGBMP) mimics a unique protective epitope on the surface of group B meningococci (GBM) and Escherichia coli K1. Using a series of monoclonal antibodies (mAbs) induced by the NPrGBMP–monomeric tetanus toxoid (TT) conjugate vaccine it was demonstrated that mAbs having specificities for both extended and conventional short segments of the NPrGBMP were formed, but only the former were bactericidal, and/or gave passive protection against live challenge by GBM. The failure of mAbs specific for short epitopes to protect was further established when (NeuPr)4–TT was used as the vaccine. Of all the mAbs produced that were specific for short internal segments of the NPrGBMP, none were protective, despite the fact that most of them cross-react with the GBM capsular polysaccharide. In contrast, most of the protective mAbs produced by NPrGBMP– TT did not recognize the group B meningococcal polysaccharide (GBMP) unless it was present in its aggregated high molecular weight form. The bactericidal epitope mimicked by the NPrGBMP was shown to be ubiquitous in the capsule of both GBM and E. coli K1 using immunogold labeling techniques and, because of its unique properties, its identification could be significant in the development of a comprehensive conjugate vaccine against group B meningococcal meningitis. This is because most known human α(2–8)-polysialic acid self-antigens can be accommodated in 30–50 α(2–8)-linked sialic acid residues, which is roughly equivalent to an 11-kD length of the GBMP. It has been hypothesized that the formation of the protective epitope on the surface of GBM is due to the interaction of helical segments of the GBMP with another molecule and that the protective epitope is mimicked by the NPrGBMP. Support for the above hypothesis is provided by the fact that the protective NPrGBMP epitope has a similar unusual length dependency to that of the GBMP epitope. PMID:9166422

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

    PubMed Central

    Nguyen, T M; Morris, G E

    1993-01-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. Images Figure 4 Figure 1 PMID:7684887

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

    SciTech Connect

    Amniai, Laziza; Lippens, Guy; Landrieu, Isabelle

    2011-09-09

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

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

    SciTech Connect

    Nguyen thi Man; Morris, G.E. )

    1993-06-01

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

  2. Widespread CD4+ T-cell reactivity to novel hTERT epitopes following vaccination of cancer patients with a single hTERT peptide GV1001.

    PubMed

    Inderberg-Suso, Else-Marit; Trachsel, Sissel; Lislerud, Kari; Rasmussen, Anne-Marie; Gaudernack, Gustav

    2012-08-01

    Understanding the basis of a successful clinical response after treatment with therapeutic cancer vaccines is essential for the development of more efficacious therapy. After vaccination with the single telomerase (hTERT) 16-mer peptide, GV1001, some patients experienced clinical responses and long-term survival. This study reports in-depth immunological analysis of the T-cell response against telomerase (hTERT) in clinically responding patients compared with clinical non-responders following vaccination with the single hTERT 16-mer peptide, GV1001. Extensive characterization of CD4+ T-cell clones specific for GV1001 generated from a lung cancer patient in complete remission after vaccination demonstrated a very broad immune response to this single peptide vaccine with differences in fine specificity, HLA restriction, affinity and function. Some CD4+ T-cell clones were cytotoxic against peptide-loaded target cells and also recognized processed recombinant hTERT protein. Furthermore, T-cell responses against several unrelated hTERT epitopes, some of which are novel, were detected, indicating extensive epitope spreading which was confirmed in other clinical responders. In contrast, patients responding immunologically, but not clinically, after vaccination did not display this intramolecular epitope spreading. Multifunctional CD4+ T-cell clones specific for novel hTERT epitopes were generated and shown to recognize a melanoma cell line. Pentamer analysis of T cells in peripheral blood also demonstrated the presence of an important CD8+ T-cell response recognizing an HLA-B7 epitope embedded in GV1001 not previously described. These results indicate that the highly diverse hTERT-specific T-cell response, integrating both T helper and CTL responses, is essential for tumor regression and the generation of long-term T-cell memory. PMID:22934259

  3. Widespread CD4+ T-cell reactivity to novel hTERT epitopes following vaccination of cancer patients with a single hTERT peptide GV1001

    PubMed Central

    Inderberg-Suso, Else-Marit; Trachsel, Sissel; Lislerud, Kari; Rasmussen, Anne-Marie; Gaudernack, Gustav

    2012-01-01

    Understanding the basis of a successful clinical response after treatment with therapeutic cancer vaccines is essential for the development of more efficacious therapy. After vaccination with the single telomerase (hTERT) 16-mer peptide, GV1001, some patients experienced clinical responses and long-term survival. This study reports in-depth immunological analysis of the T-cell response against telomerase (hTERT) in clinically responding patients compared with clinical non-responders following vaccination with the single hTERT 16-mer peptide, GV1001. Extensive characterization of CD4+ T-cell clones specific for GV1001 generated from a lung cancer patient in complete remission after vaccination demonstrated a very broad immune response to this single peptide vaccine with differences in fine specificity, HLA restriction, affinity and function. Some CD4+ T-cell clones were cytotoxic against peptide-loaded target cells and also recognized processed recombinant hTERT protein. Furthermore, T-cell responses against several unrelated hTERT epitopes, some of which are novel, were detected, indicating extensive epitope spreading which was confirmed in other clinical responders. In contrast, patients responding immunologically, but not clinically, after vaccination did not display this intramolecular epitope spreading. Multifunctional CD4+ T-cell clones specific for novel hTERT epitopes were generated and shown to recognize a melanoma cell line. Pentamer analysis of T cells in peripheral blood also demonstrated the presence of an important CD8+ T-cell response recognizing an HLA-B7 epitope embedded in GV1001 not previously described. These results indicate that the highly diverse hTERT-specific T-cell response, integrating both T helper and CTL responses, is essential for tumor regression and the generation of long-term T-cell memory. PMID:22934259

  4. 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

  5. Antibody Recognition of Shiga Toxins (Stxs): Computational Identification of the Epitopes of Stx2 Subunit A to the Antibodies 11E10 and S2C4

    PubMed Central

    Jiao, Yongjun; Legge, Fiona S.; Zeng, Xiaoyan; Treutlein, Herbert R.; Zeng, Jun

    2014-01-01

    We have recently developed a new method to predict the epitopes of the antigens that are recognized by a specific antibody. In this work, we applied the method to identify the epitopes of the Shiga toxin (Stx2 subunit A) that were bound by two specific antibodies 11E10 and S2C4. The predicted epitopes of Stx2 binding to the antibody 11E10 resembles the recognition surface constructed by the regions of Stx2 identified experimentally. For the S2C4, our results indicate that the antibody recognizes the Stx2 at two different regions on the protein surface. The first region (residues 246-254: ARSVRAVNE) is similar to the recognition region of the 11E10, while the second region is formed by two epitopes. The second region is particularly significant because it includes the amino acid sequence region that is diverse between Stx2 and other Stx (residues 176-188: QREFRQALSETAPV). This new recognition region is believed to play an important role in the experimentally observed selectivity of S2C4 to the Stx2. PMID:24516609

  6. 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

  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. 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.

  9. Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.

    PubMed

    Pasman, Yfke; Soliman, Caroline; Ramsland, Paul A; Kaushik, Azad K

    2016-09-01

    We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity. PMID:27497190

  10. CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249-59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes.

    PubMed

    Connelley, Timothy K; Li, Xiaoying; MacHugh, Niall; Colau, Didier; Graham, Simon P; van der Bruggen, Pierre; Taracha, Evans L; Gill, Andy; Morrison, William Ivan

    2016-10-01

    Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide-MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross-reactivity. The Tp249-59 and Tp250-59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide-MHC I complexes adopt structurally different conformations and Tcell receptor β sequence analysis showed that Tp249-59 and Tp250-59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant. PMID:27317384

  11. Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

    PubMed Central

    Wiley, J A; Hamel, J; Brodeur, B R

    1992-01-01

    Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report. Images PMID:1382141

  12. Recognizing child maltreatment in Bangladesh.

    PubMed

    Khan, N Z; Lynch, M A

    1997-08-01

    Concern is increasing in Bangladesh over child abuse, neglect, and exploitation. Children from all walks of life are being treated at the Child Development Center (CDC) Dhaka Shishu Hospital for neurodevelopmental problems resulting from abuse and neglect. Efforts to protect children from sexual harassment result in girls being isolated at home or married at an early age. Some young brides are eventually abandoned and forced into prostitution. Early marriage reflects the lack of acknowledgement of a period of adolescence and the belief that puberty is a marker of adulthood. Many girls aged 8-16 are employed as live-in domestic servants, and many suffer sexual as well as emotional abuse. Garment factories, on the other hand, offer girls an escape from extreme poverty, domestic service, and early marriage but are threatened by forces that condemn child labor. Rather than ending such opportunities, employers should be encouraged to provide employees with educational and welfare facilities. The CDC seeks to explore the extent and depth of the problem of child abuse while recognizing the special circumstances at work in Bangladesh. It is also necessary to raise awareness of these issues and of the discrepancies between the law and cultural practices. For example, the legal marriage age of 18 years for a woman and 21 years for a man is often ignored. Additional forms of abuse receiving the attention of women's organizations and human rights groups include the trafficking of children. A network of concerned organizations should be created to work against the child abuse, neglect, and exploitation that Bangladesh has pledged to overcome by signing the UN Convention on the Rights of the Child. PMID:9280385

  13. Producing and recognizing analogical relations.

    PubMed

    Lipkens, Regina; Hayes, Steven C

    2009-01-01

    Analogical reasoning is an important component of intelligent behavior, and a key test of any approach to human language and cognition. Only a limited amount of empirical work has been conducted from a behavior analytic point of view, most of that within Relational Frame Theory (RFT), which views analogy as a matter of deriving relations among relations. The present series of four studies expands previous work by exploring the applicability of this model of analogy to topography-based rather than merely selection-based responses and by extending the work into additional relations, including nonsymmetrical ones. In each of the four studies participants pretrained in contextual control over nonarbitrary stimulus relations of sameness and opposition, or of sameness, smaller than, and larger than, learned arbitrary stimulus relations in the presence of these relational cues and derived analogies involving directly trained relations and derived relations of mutual and combinatorial entailment, measured using a variety of productive and selection-based measures. In Experiment 1 participants successfully recognized analogies among stimulus networks containing same and opposite relations; in Experiment 2 analogy was successfully used to extend derived relations to pairs of novel stimuli; in Experiment 3 the procedure used in Experiment 1 was extended to nonsymmetrical comparative relations; in Experiment 4 the procedure used in Experiment 2 was extended to nonsymmetrical comparative relations. Although not every participant showed the effects predicted, overall the procedures occasioned relational responses consistent with an RFT account that have not yet been demonstrated in a behavior-analytic laboratory setting, including productive responding on the basis of analogies. PMID:19230515

  14. 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

  15. 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

  16. Preferential solvation: dividing surface vs excess numbers.

    PubMed

    Shimizu, Seishi; Matubayasi, Nobuyuki

    2014-04-10

    How do osmolytes affect the conformation and configuration of supramolecular assembly, such as ion channel opening and actin polymerization? The key to the answer lies in the excess solvation numbers of water and osmolyte molecules; these numbers are determinable solely from experimental data, as guaranteed by the phase rule, as we show through the exact solution theory of Kirkwood and Buff (KB). The osmotic stress technique (OST), in contrast, purposes to yield alternative hydration numbers through the use of the dividing surface borrowed from the adsorption theory. However, we show (i) OST is equivalent, when it becomes exact, to the crowding effect in which the osmolyte exclusion dominates over hydration; (ii) crowding is not the universal driving force of the osmolyte effect (e.g., actin polymerization); (iii) the dividing surface for solvation is useful only for crowding, unlike in the adsorption theory which necessitates its use due to the phase rule. KB thus clarifies the true meaning and limitations of the older perspectives on preferential solvation (such as solvent binding models, crowding, and OST), and enables excess number determination without any further assumptions. PMID:24689966

  17. Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

    PubMed Central

    Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L

    2002-01-01

    The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope. PMID:11903056

  18. Clinical spectrum associated with MOG autoimmunity in adults: significance of sharing rodent MOG epitopes.

    PubMed

    Sepúlveda, Maria; Armangue, Thaís; Martinez-Hernandez, Eugenia; Arrambide, Georgina; Sola-Valls, Nuria; Sabater, Lidia; Téllez, Nieves; Midaglia, Luciana; Ariño, Helena; Peschl, Patrick; Reindl, Markus; Rovira, Alex; Montalban, Xavier; Blanco, Yolanda; Dalmau, Josep; Graus, Francesc; Saiz, Albert

    2016-07-01

    The aim of this study was to report the clinical spectrum associated with antibodies to myelin oligodendrocyte glycoprotein (MOG) in adult patients, and to assess whether phenotypic variants are dependent on recognition of rodent MOG epitopes. We retrospectively analyzed the features, course and outcome of 56 patients whose samples were investigated by brain tissue immunohistochemistry and cell-based assays using human and rodent MOG. The median age at symptom onset was 37 years (range 18-70); 35 patients (63 %) were female. After a median follow-up of 43 months (range 4-554), only 14 patients (25 %) developed a neuromyelitis optica spectrum disorder (NMOSD), 27 patients (47 %) retained the initial diagnosis of isolated optic neuritis, 7 (12 %) of longitudinally extensive transverse myelitis, and 2 (4 %) of acute disseminated encephalomyelitis; 6 patients (11 %) developed atypical demyelinating syndromes (4 had relapsing episodes of short myelitis lesions which in one occurred with optic neuritis; 1 had relapsing brainstem symptoms, and 1 relapsing demyelinating encephalomyelitis). The course was frequently associated with relapses (71 %) and good outcome. Twenty-seven patients (49 %) had antibodies that recognized rodent MOG epitopes, and 9 of them (16 %) showed a myelin staining pattern in rodent tissue. Only the myelin staining pattern was linked to NMOSD (p = 0.005). In conclusion, MOG autoimmunity in adult patients associates with a clinical spectrum wider than the one expected for patients with suspected NMOSD and overall good outcome. Antibodies to rodent MOG epitopes do not associate with any phenotypic variant. PMID:27147513

  19. Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.

    PubMed

    Masri, Heather P; Cornelissen, Cynthia Nau

    2002-02-01

    The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed. PMID:11796606

  20. The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

    PubMed

    Carrabino, D; D'Onorio De Meo, P; Sanna, N; Castrignanò, T; Orsini, M; Floris, M; Tramontano, A

    2007-06-01

    For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface. PMID:17695751

  1. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection.

    PubMed

    Deng, Kai; Liu, Ruyu; Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1-6 was determined. We defined a domain comprising amino acids (aa) 192-221, 232-251, 262-281 and 292-331 of E1, and 421-543, 564-583, 594-618 and 634-673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444-463) and PUHI45 (aa 604-618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  2. Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

    PubMed

    Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L

    2002-04-01

    The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope. PMID:11903056

  3. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

    PubMed Central

    Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  4. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  5. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    SciTech Connect

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    2011-10-01

    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

  6. 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

  7. Synthesis of Monocrystalline Nanoframes of Prussian Blue Analogues by Controlled Preferential Etching.

    PubMed

    Zhang, Wei; Zhao, Yanyi; Malgras, Victor; Ji, Qingmin; Jiang, Dongmei; Qi, Ruijuan; Ariga, Katsuhiko; Yamauchi, Yusuke; Liu, Jian; Jiang, Ji-Sen; Hu, Ming

    2016-07-11

    Metal cyanide coordination compounds are recognized as promising candidates for broad applications because of their tailorable and adjustable frameworks. Developing the nanostructure of a coordination compound may be an effective way to enhance the performance of that material in application-based roles. A controllable preferential etching method is described for synthesis of monocrystalline Prussian blue analogue (PBA) nanoframes, without the use of organic additives. The PBA nanoframes show remarkable rate performance and cycling stability for sodium/lithium ion insertion/extraction. PMID:27355859

  8. Endolysins of Bacillus anthracis Bacteriophages Recognize Unique Carbohydrate Epitopes of Vegetative Cell Wall Polysaccharides with High Affinity and Selectivity

    PubMed Central

    Mo, Kai-For; Li, Xiuru; Li, Huiqing; Low, Lieh Yoon; Quinn, Conrad P.

    2012-01-01

    Bacteriophages express endolysins which are the enzymes that hydrolyze peptidoglycan resulting in cell lysis and release of bacteriophages. Endolysins have acquired stringent substrate specificities, which have been attributed to cell wall binding domains (CBD). Although it has been realized that CBDs of bacteriophages that infect Gram-positive bacteria target cell wall carbohydrate structures, molecular mechanisms that confer selectivity are not understood. A range of oligosaccharides, derived from the secondary cell wall polysaccharides of Bacillus anthracis, has been chemically synthesized. The compounds contain an α-D-GlcNAc-(1→4)-β-D-ManNAc-(1→4)-β-D-GlcNAc backbone that is modified by various patterns of α-D-Gal and β-D-Gal branching points. The library of compounds could readily be prepared by employing a core trisaccharide modified by the orthogonal protecting groups Nα-9-fluorenylmethyloxycarbonate (Fmoc), 2-methylnaphthyl ether (Nap) and levulinoyl ester (Lev) and dimethylthexylsilyl ether (TDS) at key branching points. Dissociation constants for the binding the cell wall binding domains of the endolysins PlyL and PlyG were determined by surface plasmon resonance (SPR). It was found that the pattern of galactosylation greatly influenced binding affinities, and in particular a compound having a galactosyl moiety at C-4 of the non-reducing GlcNAc moiety bound in the low micromolar range. It is known that secondary cell wall polysaccharides of various bacilli may have both common and variable structural features and in particular differences in the pattern of galactosylation have been noted. Therefore, it is proposed that specificity of endolysins for specific bacilli is achieved by selective binding to a uniquely galactosylated core structure. PMID:22935003

  9. Epitope focusing in the primary cytotoxic T cell response to Epstein-Barr virus and its relationship to T cell memory.

    PubMed

    Steven, N M; Leese, A M; Annels, N E; Lee, S P; Rickinson, A B

    1996-11-01

    The relationship between primary and memory cytotoxic T lymphocyte (CTL) responses, and the factors influencing entry into memory, are poorly understood. Here we address this in the context of Epstein-Barr virus (EBV), a persistent human herpesvirus in which memory CTL responses in long-term virus carriers are highly focused on epitopes preferentially drawn from just three of the eight available virus latent proteins, EBNAs 3A, 3B, and 3C. To determine whether this unusual level of focusing is a consequence of long-term virus challenge, we carried out a detailed analysis of EBV antigen/epitope specificities in the primary virus-induced CTL response in 10 infectious mononucleosis (IM) patients of different HLA types. Primary effectors, studied in ex vivo assays and by limiting dilution cloning in vitro, were again highly skewed toward a small number of viral epitopes, almost all derived from the EBNA3 proteins, with CTL to the immunodominant epitope accounting for at least 1% of the circulating CD8+ IM T cell pool. This is the first unequivocal demonstration of an EBV-specific CD8+ CTL response in IM. Prospective studies on individual patients showed that, whereas all of the EBV reactivities found in CTL memory had been detectable earlier during primary infection, the memory population was not simply a scaled down version of the primary response. In particular (a) differences in the relative frequencies of CTL to immunodominant versus subdominant epitopes appeared to be much less marked in memory than in primary populations, and (b) we found at least one clear example in which a significant virus-specific reactivity within the primary response was never detectable in memory. PMID:8920868

  10. Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

    PubMed Central

    Borthwick, Nicola; Ahmed, Tina; Ondondo, Beatrice; Hayes, Peter; Rose, Annie; Ebrahimsa, Umar; Hayton, Emma-Jo; Black, Antony; Bridgeman, Anne; Rosario, Maximillian; Hill, Adrian VS; Berrie, Eleanor; Moyle, Sarah; Frahm, Nicole; Cox, Josephine; Colloca, Stefano; Nicosia, Alfredo; Gilmour, Jill; McMichael, Andrew J; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. PMID:24166483

  11. Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies.

    PubMed

    Cheng, Hsien-Jen; Lin, Chiou-Feng; Lei, Huan-Yao; Liu, Hsiao-Sheng; Yeh, Trai-Ming; Luo, Yueh-Hsia; Lin, Yee-Shin

    2009-01-01

    We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope. PMID:18997103

  12. 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.

  13. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors.

    PubMed

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-07-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8(+) T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8(+) T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8(+) T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8(+) T-cell clones are highly focused on their index peptide sequence and that 'CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8(+) T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  14. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  15. 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

  16. 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

  17. Generation of monoclonal antibodies to the specific sugar epitopes of Mycobacterium avium complex serovars.

    PubMed Central

    Rivoire, B; Ranchoff, B J; Chatterjee, D; Gaylord, H; Tsang, A Y; Kolk, A H; Aspinall, G O; Brennan, P J

    1989-01-01

    Monoclonal antibodies have been generated to the unique distal sugar epitopes on the oligosaccharide haptens of the glycopeptidolipid antigens of clinically prominent members of the Mycobacterium avium serocomplex. Thus, antibodies are described that recognize the distal O-acetyl-alpha-L-rhamnopyranosyl residue of the specific glycopeptidolipid of M. avium serovar 1, the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranose of serovar 2, the 4-O-methyl-alpha-L-rhamnopyranosyl-(1----4)-2-O-methyl-alpha-L- fucopyranosyl unit of serovar 4, the 4,6-(1'-carboxyethylidene)-3-O-methyl-beta-D-glucopyranosyl unit of serovar 8 [and the 4,6-(1'-carboxyethylidene)-beta-D-glucopyranosyl residue of serovar 21], and the 4-O-acetyl-2,3-di-O-methyl-alpha-L-fucopyranosyl-(1----4)-beta-D- glucuronopyranosyl unit of serovar 9. Epitope definition was arrived at through use of the pure, chemically defined glycopeptidolipid antigens and neoglycoproteins containing the chemically synthesized distal sugars of some select serovars. These monoclonal antibodies combined with the already published information on the structure of the antigen determinants and the tools used to arrive at these structures provide powerful means for fundamental studies on the role of these antigens in immunopathogenesis and for the precise mapping of the epidemiology of opportunistic infections caused by M. avium. Images PMID:2476400

  18. Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP)

    PubMed Central

    PéRez De La Lastra, J M; Van Den Berg, C W; Bullido, R; Almazán, F; Domínguez, J; Llanes, D; Morgan, B P

    1999-01-01

    Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals – a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I. PMID:10233756

  19. Innate sensing of oxidation-specific epitopes in health and disease.

    PubMed

    Binder, Christoph J; Papac-Milicevic, Nikolina; Witztum, Joseph L

    2016-08-01

    Ageing, infections and inflammation result in oxidative stress that can irreversibly damage cellular structures. The oxidative damage of lipids in membranes or lipoproteins is one of these deleterious consequences that not only alters lipid function but also leads to the formation of neo-self epitopes - oxidation-specific epitopes (OSEs) - which are present on dying cells and damaged proteins. OSEs represent endogenous damage-associated molecular patterns that are recognized by pattern recognition receptors and the proteins of the innate immune system, and thereby enable the host to sense and remove dangerous biological waste and to maintain homeostasis. If this system is dysfunctional or overwhelmed, the accumulation of OSEs can trigger chronic inflammation and the development of diseases, such as atherosclerosis and age-related macular degeneration. Understanding the molecular components and mechanisms that are involved in this process will help to identify individuals with an increased risk of developing chronic inflammation, and will also help to indicate novel modes of therapeutic intervention. PMID:27346802

  20. Maturation-Induced Cloaking of Neutralization Epitopes on HIV-1 Particles

    PubMed Central

    Joyner, Amanda S.; Willis, Jordan R.; Crowe, James E.; Aiken, Christopher

    2011-01-01

    To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER. PMID:21931551

  1. Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

    PubMed

    Doorbar, J; Ely, S; Coleman, N; Hibma, M; Davies, D H; Crawford, L

    1992-03-01

    The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer. PMID:1371027

  2. [Preparation of monoclonal antibodies against enterovirus type 71 with an epitope-incorporated adenovirus type 3 vector].

    PubMed

    Fan, Ye; Tian, Xingui; Xue, Chunyan; Liu, Minglong; Zhou, Zhichao; Li, Xiao; Li, Chenyang; Zhou, Rong

    2016-08-01

    Objective To develop the monoclonal antibodies (mAbs) against enterovirus type 71 (EV71). Methods Two neutralization epitopes, SP70 and SP55, from EV71 were cloned into the hexon gene of adenovirus type 3 to generate a recombinant adenovirus type 3 (R1R2A3) presenting SP70 and SP55 antigens. BALB/c mice were immunized with the R1R2A3. The mAbs were developed with hybridoma technology and were analyzed with microneutralizing assay, indirect ELISA, Western blotting and direct immunofluorescence assay (DFA). Results The study obtained four hybridoma cell clones, 2C4, D2C9, I2G2 and I12C3. ELISA showed that the titer of D2C9 against EV71 was 1:8 000 000 and the titers of 2C4, I2G2, and I12C3 all were 1:500 000. ELISA and Western blotting demonstrated that all mAbs could specifically recognize the VP1 of EV71. In addition, D2C9 recognized the SP70 epitope, and 2C4, I12C3 and I2G2 all recognized the SP55 epitope. DFA revealed that all mAbs could react with EV71, but not with Coxsackie virus A16 (CoxA16). Conclusion Four mAbs against EV71 have been developed successfully, and all of them could react with EV71 rather than CoxA16. PMID:27412945

  3. 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

  4. Reversible conformational change of tau2 epitope on exposure to detergent in glial cytoplasmic inclusions of multiple system atrophy.

    PubMed

    Shibuya, Katsuhiko; Uchihara, Toshiki; Nakamura, Ayako; Ishiyama, Miyako; Yamaoka, Keiko; Yagishita, Saburo; Iwabuchi, Kiyoshi; Kosaka, Kenji

    2003-05-01

    Tau-like immunoreactivity (IR) on glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) was investigated with a panel of anti-tau antibodies and we found that tau2, one of the phosphorylation-independent antibodies, preferentially immunolabeled GCIs. Co-presence (0.03%) of polyethyleneglycol- p-isooctylphenyl ether (Triton X-100, TX) with tau2, however, abolished this IR on GCIs, but did not abolish tau2 IR on neurofibrillary tangles (NFTs). Tau2-immunoreactive bands on immunoblot of brain homogenates from MSA brains were retrieved mainly in a TRIS-saline-soluble fraction, as reported in normal brains. This was in contrast to SDS-soluble fractions from brain with Down's syndrome, which contained tau2-immunoreactive bands of higher molecular weight. It indicates that the appearance of tau2 IR on GCIs is not related to hyperphosphorylation of tau. These tau2-immunoreactive bands, except those from bovine brain, were similarly abolished in the presence of TX (0.06%), and repeated washing after exposure to TX restored the tau2 IR on immunohistochemistry and on immunoblot. These findings can be explained if the modified tau2 epitope undergoes a reversible conformational change on exposure to TX, which is reversible after washing. Because the conformation centered at Ser101 of bovine tau is crucial for its affinity to tau2, the Ser-like conformation mimicked by its human counterpart Pro may represent pathological modification of tau shared by GCIs and NFTs. The relative resistance of tau2 epitope on NFTs on exposure to TX suggests that tau woven into NFTs confers additional stability to the pathological conformation of tau2 epitope. The conformation of the tau2 epitope in GCIs is not as stable as in NFTs, suggesting that tau proteins are not the principal constituents of the fibrillary structures of GCIs, even though they were immunodecorated with tau2. The difference in the susceptibility of the tau2 epitope to TX may distinguish its conformational states

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

    PubMed

    Han, Youngshin; Lin, Jing; Bardina, Ludmilla; Grishina, Galina A; Lee, Chaeyoon; Seo, Won Hee; Sampson, Hugh A

    2016-01-01

    Ara h 2, a peanut 2S albumin, is associated with severe allergic reactions, but a homologous protein, soybean 2S albumin, is not recognized as an important allergen. Structural difference between these proteins might explain this clinical discrepancy. Therefore, we mapped sequential epitopes and compared the structure of Ara h 2, Soy Al 1, and Soy Al 3 (Gly m 8) to confirm whether structural differences account for the discrepancy in clinical responses to these two proteins. Commercially synthesized peptides covering the full length of Ara h 2 and two soybean 2S albumins were analyzed by peptide microarray. Sera from 10 patients with peanut and soybean allergies and seven non-atopic controls were examined. The majority of epitopes in Ara h 2 identified by microarray are consistent with those identified previously. Several regions in the 2S albumins are weakly recognized by individual sera from different patients. A comparison of allergenic epitopes on peanut and soybean proteins suggests that loop-helix type secondary structures and some amino acids with a large side chain including lone electron pair, such as arginine, glutamine, and tyrosine, makes the peptides highly recognizable by the immune system. By utilizing the peptide microarray assay, we mapped IgE epitopes of Ara h 2 and two soybean 2S albumins. The use of peptide microarray mapping and analysis of the epitope characteristics may provide critical information to access the allergenicity of food proteins. PMID:27187334

  6. Preferential Option for the Poor: Making a Pedagogical Choice

    ERIC Educational Resources Information Center

    Kirylo, James D.

    2006-01-01

    When children are sick, hurt, or in desperate need, parents/caregivers naturally make preferential options for them. Yet, as it relates to social justice, particularly when working with students from marginalized and poverty situations, the concept of making a preferential option in a school setting is not as clear. However, a school setting is a…

  7. 19 CFR 10.213 - Articles eligible for preferential treatment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Articles eligible for preferential treatment. 10...; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. Textile and Apparel Articles Under the African Growth and Opportunity Act § 10.213 Articles eligible for preferential...

  8. 19 CFR 10.213 - Articles eligible for preferential treatment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Articles eligible for preferential treatment. 10...; DEPARTMENT OF THE TREASURY ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. Textile and Apparel Articles Under the African Growth and Opportunity Act § 10.213 Articles eligible for preferential...

  9. 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

  10. Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

    PubMed Central

    Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

    2013-01-01

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

  11. Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein

    SciTech Connect

    Zhou Jian; Blissard, Gary W. . E-mail: gwb1@cornell.edu

    2006-09-01

    The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the endosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function normally. However, an anti-c-Myc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64.

  12. Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants.

    PubMed

    Li, Min; Wang, Xin; Cao, Lu; Lin, Zhijie; Wei, Minxi; Fang, Mujin; Li, Shaowei; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2016-08-17

    Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples. PMID:27426626

  13. 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

  14. The Transport Signal on Sec22 for Packaging into COPII-Coated Vesicles is a Conformational Epitope

    SciTech Connect

    Mancias,J.; Goldberg, J.

    2007-01-01

    The mechanism of cargo concentration into ER-derived vesicles involves interactions between the COPII vesicular coat complex and cargo transport signals-peptide sequences of 10-15 residues. The SNARE protein Sec22 contains a signal that binds the COPII subcomplex Sec23/24 and specifies its endoplasmic reticulum (ER) exit as an unassembled SNARE. The 200 kDa crystal structure of Sec22 bound to Sec23/24 reveals that the transport signal is a folded epitope rather than a conventional short peptide sequence. The NIE segment of the SNARE motif folds against the N-terminal longin domain, and this closed form of Sec22 binds at the Sec23/24 interface. Thus, COPII recognizes unassembled Sec22 via a folded epitope, whereas Sec22 assembly into SNARE complexes would mask the NIE segment. The concept of a conformational epitope as a transport signal suggests packaging mechanisms in which a coat is sensitive to the folded state of a cargo protein or the assembled state of a multiprotein complex.

  15. Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes

    PubMed Central

    Tellam, Judy; Fogg, Mark H.; Rist, Michael; Connolly, Geoff; Tscharke, David; Webb, Natasha; Heslop, Lea; Wang, Fred; Khanna, Rajiv

    2007-01-01

    A significant proportion of endogenously processed CD8+ T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8+ T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus–encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1ΔGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1ΔGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1ΔGA. As a consequence, a higher number of major histocompatibility complex–peptide complexes can be detected on the surface of cells expressing EBNA1ΔGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8+ T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins. PMID:17312009

  16. Identification of the epitope for a monoclonal antibody that blocks platelet aggregation induced by type III collagen.

    PubMed Central

    Glattauer, V; Werkmeister, J A; Kirkpatrick, A; Ramshaw, J A

    1997-01-01

    A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with alpha2beta1-integrin of platelets. PMID:9173900

  17. Anti-H1 histone antibodies in systemic lupus erythematosus: epitope localization after immunoblotting of chymotrypsin-digested H1.

    PubMed Central

    Costa, O; Tchouatcha-Tchouassom, J C; Roux, B; Monier, J C

    1986-01-01

    Using micro enzyme-linked immunosorbent assay (micro-ELISA) anti-H1 antibodies are most frequently seen in systemic lupus erythematosus (SLE) (61.4% of patients). Positive anti-H1 ELISA reactions are rare in rheumatoid arthritis (RA) (5.8% of cases), melanomas (16.7%), leukaemias (13.6%) and other cancers (5.6%). In SLE, the immunoglobulins (Ig) which constitute anti-H1 antibodies are, by order of importance, IgM, IgG and IgA. By means of immunoblotting using H1 solutions digested by alpha-1-chymotrypsin fixed on collagen membranes, we have shown that all the SLE sera containing anti-H1 antibodies recognize the sequential epitopes that are found on the carboxy terminal tail and, for 28% of anti-H1 sera, also the epitopes present on the aminoterminal half. The technique used did not make it possible to determine with certainty whether anti-H1 autoantibodies are also directed against the conformational epitopes of the globular part of the molecule. Images Fig. 2 PMID:2423279

  18. Invertebrate host-parasite relationships: convergent evolution of a tropomyosin epitope between Schistosoma sp., Fasciola hepatica, and certain pulmonate snails.

    PubMed

    Weston, D; Allen, B; Thakur, A; LoVerde, P T; Kemp, W M

    1994-05-01

    Monoclonal antibodies (mAb) directed against Schistosoma mansoni tropomyosin isoform, SMTM (Xu et al. Experimental Parasitology 69, 373-392, 1989), were used to test for cross-reactivity with Biomphalaria glabrata antigens. One mAb (1F10) recognized antigens of 39, 41, and 80 kDa in a snail head/foot antigen preparation but not a hepatopancreas antigen preparation. Another mAb (1C1) cross-reacted with a 39-kDa antigen in the head/foot extract but not in the hepatopancreas extract. Epitope mapping revealed the 1F10 epitope to be between amino acids 135 and 188 of both Bg39 (Dissous et al. Molecular and Biochemical Parasitology 43, 245-256, 1990) and BgTMII (Weston and Kemp, Experimental Parasitology 76, 358-370, 1993), while the 1C1 epitope was located between amino acids 189 and 213 of BgTMII. Various invertebrate species, including members from Trematoda, Pulmonata, Annelida, and Arthropoda, were tested for cross-reactivity with the monoclonal antibodies. While the 1F10 mAb displayed broad invertebrate cross-reactivity, the 1C1 mAb cross-reactivity was restricted to schistosomes, F. hepatica, and the pulmonate snails B. glabrata and Physa sp. PMID:7512930

  19. 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.

  20. 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

  1. Characterization of Puumala virus nucleocapsid protein: identification of B-cell epitopes and domains involved in protective immunity.

    PubMed

    Lundkvist, A; Kallio-Kokko, H; Sjölander, K B; Lankinen, H; Niklasson, B; Vaheri, A; Vapalahti, O

    1996-02-15

    B-cell epitopes in the nucleocapsid protein (N) of Puumala (PUU) virus were investigated by use of truncated recombinant proteins and overlapping peptides. Six of seven epitopes, recognized by bank vole monoclonal antibodies, were localized within the amino-terminal region of the protein (aa 1-79). Polyclonal antibodies from wild-trapped or experimentally infected bank voles identified epitopes located over the entire protein. Antibody end-point titers to different N fragments indicated that the amino-terminal region is the major antigenic target in PUU virus-infected bank voles. To investigate the role of PUU virus N in protective immunity, we analyzed the immunogenicity of truncated recombinant N and developed an animal model based on colonized bank voles. No PUU virus N antigen, nor any glycoprotein-specific antibodies, could be detected after virus challenge in animals immunized with an amino-terminal fragment (aa 1-118), a fragment covering two thirds of the animals immunized with shorter N fragments displayed either N antigen, or glycoprotein-specific antibodies, suggestive of partial protection. Prechallenge sera from all groups of immunized animals were found negative or only weakly positive for neutralizing antibodies when assayed by focus reduction neutralization test, which indicated an important role for cell-mediated immunity in protection. PMID:8607269

  2. 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

  3. 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

  4. 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

  5. Preferentially Cytotoxic Constituents of Andrographis paniculata and their Preferential Cytotoxicity against Human Pancreatic Cancer Cell Lines.

    PubMed

    Lee, Sullim; Morita, Hiroyuki; Tezuka, Yasuhiro

    2015-07-01

    In the course of our search for anticancer agents based on a novel anti-austerity strategy, we found that the 70% EtOH extract of the crude drug Andrographis Herba (aerial parts of Andrographis paniculata), used in Japanese Kampo medicines, killed PANC-1 human pancreatic cancer cells preferentially in nutrient-deprived medium (NDM). Phytochemical investigation of the 70% EtOH extract led to the isolation of 21 known compounds consisting of six labdane-type diterpenes (11, 15, 17-19, 21), six flavones (5, 7, 10, 12, 14, 20), three flavanones (2, 6, 16), two sterols (3, 8), a fatty acid (1), a phthalate (4), a triterpene (9), and a monoterpene (13). Among them, 14-deoxy-11,12-didehydroandrographolide (17) displayed the most potent preferential cytotoxicity against PANC-1 and PSN-1 cells with PC50 values of 10.0 μM and 9.27 μM, respectively. Microscopical observation, double staining with ethidium bromide (EB) and acridine orange (AO), and flow cytometry with propidium iodide/annexin V double staining indicated that 14-deoxy-11,12-didehydroandrographolide (17) triggered apoptosis-like cell death in NDM with an amino acids and/or serum-sensitive mode. PMID:26410998

  6. Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.

    PubMed Central

    Luther, T.; Magdolen, V.; Albrecht, S.; Kasper, M.; Riemer, C.; Kessler, H.; Graeff, H.; Müller, M.; Schmitt, M.

    1997-01-01

    uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface. As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods. Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen. The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized. All MAbs reacted under reducing conditions in immunoblot analyses with E. coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells. Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well. By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best. Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR). In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors. Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA. As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7

  7. 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.

  8. 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

  9. Human B-cell epitopes of Puumala virus nucleocapsid protein, the major antigen in early serological response.

    PubMed

    Vapalahti, O; Kallio-Kokko, H; Närvänen, A; Julkunen, I; Lundkvist, A; Plyusnin, A; Lehväslaiho, H; Brummer-Korvenkontio, M; Vaheri, A; Lankinen, H

    1995-08-01

    Puumala virus (PUU) is a member of the Hantavi rus genus in the family Bunyaviridae and the etiologic agent of nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome (HFRS). In this study we compared the immunofluorescence patterns of NE sera and antibodies raised against recombinant PUU proteins and confirm that the nucleocapsid protein is the major target in the early IgG response of NE patients and provides the molecular basis for simple and rapid differentiation between acute illness and old immunity by granular vs. diffuse fluorescence staining in the indirect immunofluorescence test. The differential kinetics of B-cell responses to PUU nucleocapsid vs. envelope proteins was emphasized further by the endpoint titres of IgG antibodies to N, G1 and G2 proteins in NE patients. The granular fluorescence correlated with low IgG avidity in 99.8%, and diffuse fluorescence with high avidity in 100% of 617 NE sera studied. Epitope scanning with overlapping 14-mer peptides covering the whole nucleocapsid protein by a shift of 3 amino acids revealed six major antigenic epitopes recognized by sera from acute-phase NE patients. The epitopes clustered mainly in the hydrophilic regions, and two of them in a highly variable region which could probably serve as an antigen to distinguish serologically between infections of closely related hantaviruses, some apparently apathogenic, some causing lethal infections. The anti-peptide epitope pattern varied between different individuals and a collection of several pin-bound peptides was needed to be recognised by most NE sera studied. PMID:7595404

  10. 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

  11. Preferential Interactions and the Effect of Protein PEGylation

    PubMed Central

    Holm, Louise Stenstrup; Thulstrup, Peter W.; Kasimova, Marina R.; van de Weert, Marco

    2015-01-01

    Background PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of protein drugs, whereas formulation excipients are used for stabilization of proteins during storage. Here we investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein. Methodology/Principal Findings The model protein hen egg white lysozyme was doubly PEGylated on two lysines with 5 kDa linear PEGs (mPEG-succinimidyl valerate, MW 5000) and studied in the absence and presence of preferentially excluded sucrose and preferentially bound guanine hydrochloride. Structural characterization by far- and near-UV circular dichroism spectroscopy was supplemented by investigation of protein thermal stability with the use of differential scanning calorimetry, far and near-UV circular dichroism and fluorescence spectroscopy. It was found that PEGylated lysozyme was stabilized by the preferentially excluded excipient and destabilized by the preferentially bound excipient in a similar manner as lysozyme. However, compared to lysozyme in all cases the melting transition was lower by up to a few degrees and the calorimetric melting enthalpy was decreased to half the value for PEGylated lysozyme. The ratio between calorimetric and van’t Hoff enthalpy suggests that our PEGylated lysozyme is a dimer. Conclusion/Significance The PEGylated model protein displayed similar stability responses to the addition of preferentially active excipients. This suggests that formulation principles using preferentially interacting excipients are similar for PEGylated and non-PEGylated proteins. PMID:26230338

  12. Soil properties and preferential solute transport at the field scale

    NASA Astrophysics Data System (ADS)

    Koestel, J. K.; Luong, N. M.; Nørgaard, T.; Vendelboe, A. L.; Moldrup, P.; Jarvis, N. J.; Lamandé, M.; Iversen, B. V.; Wollesen de Jonge, L.

    2012-04-01

    An important fraction of water flow and solute transport through soil takes place through preferential flow paths. Although this had been already observed in the nineteenth century, it had been forgotten by the scientific community until it was rediscovered during the 1970s. The awareness of the relevance of preferential flow was broadly re-established in the community by the early 1990s. However, since then, the notion remains widespread among soil scientists that the occurrence and strength of preferential flow cannot be predicted from measurable proxy variables such as soil properties or land management practices (e.g. Beven, K., 1991, Modeling preferential flow - an uncertain future, Preferential Flow, 1-11). In our study, we present evidence that disproves this notion. We evaluated breakthrough curve experiments under a constant irrigation rate of 1 cm/h conducted on 65 soil columns (20 cm diameter and 20 height) which had been sampled from an approximately 1 ha large loamy field-site in Silstrup, Denmark. We show that the holdback factor, which is an indicator for the strength of preferential transport, is strongly correlated to the bulk density, which in turn is correlated to the organic matter content. By applying multiple linear regression in a bootstrapping framework, we could estimate the holdback factor from the bulk density and the very fine sand fraction with a coefficient of determination of 0.65. Our results raise hopes that it is indeed possible to establish pedotransfer functions for soil susceptibility to preferential flow and transport.

  13. 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

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

    PubMed

    Hoffmann, Thomas; Krackhardt, Angela M; Antes, Iris

    2015-07-01

    T-cell receptors (TCR) play an important role in the adaptive immune system as they recognize pathogen- or cancer-based epitopes and thus initiate the cell-mediated immune response. Therefore there exists a growing interest in the optimization of TCRs for medical purposes like adoptive T-cell therapy. However, the molecular mechanisms behind T-cell signaling are still predominantly unknown. For small sets of TCRs it was observed that the angle between their Vα- and Vβ-domains, which bind the epitope, can vary and might be important for epitope recognition. Here we present a comprehensive, quantitative study of the variation in the Vα/Vβ interdomain-angle and its influence on epitope recognition, performing a systematic bioinformatics analysis based on a representative set of experimental TCR structures. For this purpose we developed a new, cuboid-based superpositioning method, which allows a unique, quantitative analysis of the Vα/Vβ-angles. Angle-based clustering led to six significantly different clusters. Analysis of these clusters revealed the unexpected result that the angle is predominantly influenced by the TCR-clonotype, whereas the bound epitope has only a minor influence. Furthermore we could identify a previously unknown center of rotation (CoR), which is shared by all TCRs. All TCR geometries can be obtained by rotation around this center, rendering it a new, common TCR feature with the potential of improving the accuracy of TCR structure prediction considerably. The importance of Vα/Vβ rotation for signaling was confirmed as we observed larger variances in the Vα/Vβ-angles in unbound TCRs compared to epitope-bound TCRs. Our results strongly support a two-step mechanism for TCR-epitope: First, preformation of a flexible TCR geometry in the unbound state and second, locking of the Vα/Vβ-angle in a TCR-type specific geometry upon epitope-MHC association, the latter being driven by rotation around the unique center of rotation. PMID:26185983

  15. Duality between preferential attachment and static networks on hyperbolic spaces

    NASA Astrophysics Data System (ADS)

    Ferretti, L.; Cortelezzi, M.; Mamino, M.

    2014-02-01

    There is a complex relation between the mechanism of preferential attachment, scale-free degree distributions and hyperbolicity in complex networks. In fact, both preferential attachment and hidden hyperbolic spaces often generate scale-free networks. We show that there is actually a duality between a class of growing spatial networks based on preferential attachment on the sphere and a class of static random networks on the hyperbolic plane. Both classes of networks have the same scale-free degree distribution as the Barabasi-Albert model. As a limit of this correspondence, the Barabasi-Albert model is equivalent to a static random network on an hyperbolic space with infinite curvature.

  16. The TAG family of cancer/testis antigens is widely expressed in a variety of malignancies and gives rise to HLA-A2-restricted epitopes.

    PubMed

    Adair, Sara J; Carr, Tiffany M; Fink, Mitsú J; Slingluff, Craig L; Hogan, Kevin T

    2008-01-01

    The TAG-1, TAG-2a, TAG-2b, and TAG-2c cancer/testis genes, known to be expressed in an unusually high percentage of melanoma cell lines, are shown here to be expressed in a variety of tumor lines of diverse histologic type, including cancers of the brain, breast, colon, lung, ovary, pharynx, and tongue. The genes are also expressed in fresh, uncultured melanoma, and ovarian cancer cells. Epitope prediction algorithms were used to identify potential HLA-A1, HLA-A2, HLA-A3, HLA-B7, and HLA-B8 epitopes, and these potential epitopes were tested for their ability to stimulate a peptide-specific cytotoxic T lymphocyte response using lymphocytes from healthy donors. Two HLA-A2-restricted epitopes (SLGWLFLLL and LLLRLECNV) were identified using this approach. Cytotoxic T lymphocytes specific for each of these peptides were capable of recognizing tumor cells expressing both the corresponding class I major histocompatibility complex encoded molecule and the TAG genes. These results indicate that TAG-derived peptides may be good components of a therapeutic vaccine designed to target melanoma and a variety of epithelial cell-derived malignancies. PMID:18157007

  17. 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

  18. Human monoclonal antibodies isolated from type I diabetes patients define multiple epitopes in the protein tyrosine phosphatase-like IA-2 antigen.

    PubMed

    Kolm-Litty, V; Berlo, S; Bonifacio, E; Bearzatto, M; Engel, A M; Christie, M; Ziegler, A G; Wild, T; Endl, J

    2000-10-15

    Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2beta-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777-979. Two of these hAbs cross-reacted with the related IA-2beta PTP-like domain (IA-2beta aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687-979) for binding, but critical amino acids clustered in the N-terminal region 687-777. An additional epitope could be localized within the juxtamembrane domain (aa 603-779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a V(H) germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven. PMID:11035111

  19. 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.

  20. Vitrification: Machines learn to recognize glasses

    NASA Astrophysics Data System (ADS)

    Ceriotti, Michele; Vitelli, Vincenzo

    2016-05-01

    The dynamics of a viscous liquid undergo a dramatic slowdown when it is cooled to form a solid glass. Recognizing the structural changes across such a transition remains a major challenge. Machine-learning methods, similar to those Facebook uses to recognize groups of friends, have now been applied to this problem.

  1. The Developmental Dimensions of Recognizing Racist Thoughts

    ERIC Educational Resources Information Center

    Torres, Vasti

    2009-01-01

    This study focuses on understanding the developmental process that occurs when racist ideas are recognized as a part of college students' thought processes. Longitudinal data were collected from 29 Latino/a college students in order to illustrate how these students made meaning of racist thoughts when they began to recognize it. The framework of…

  2. Higher-Order Neural Networks Recognize Patterns

    NASA Technical Reports Server (NTRS)

    Reid, Max B.; Spirkovska, Lilly; Ochoa, Ellen

    1996-01-01

    Networks of higher order have enhanced capabilities to distinguish between different two-dimensional patterns and to recognize those patterns. Also enhanced capabilities to "learn" patterns to be recognized: "trained" with far fewer examples and, therefore, in less time than necessary to train comparable first-order neural networks.

  3. Teaching Students to Recognize Irony

    ERIC Educational Resources Information Center

    Milner, Joseph O.; Hawkins, Robin H.; Milner, Lucy M.

    2014-01-01

    This article exposes the problem of using declarative rather than procedural knowledge to help K--12 students recognize irony in stories. It offers commonplace procedures drawn from students' everyday language experience together with more abstract irony clues to help students recognize irony in stories and increase their story comprehension.…

  4. 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

  5. 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

  6. Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame

    PubMed Central

    Rutkowski, Melanie R.; Ho, On; Green, William R.

    2009-01-01

    The biological significance of protective CD8 T-cell-mediated responses against non-traditional alternative reading frame epitopes remains relatively unknown. Cytolytic CD8 T cells (CTL) specific for a non-traditional cryptic MHC class I epitope, SYNTGRFPPL, are critically involved in the protection of mice during infection with the LP-BM5 murine retrovirus. The goal of this study was to determine the functional properties of the protective SYNTGRFPPL-specific CTL during LP-BM5 infection of susceptible BALB/c CD8−/− mice. Direct infection experiments and adoptive transfer of CD8 T cells derived from perforin (pfp)−/−, IFNγ−/−, FasL−/− and, as a positive control, wild-type BALB/c mice, were utilized to assess the effector mechanisms responsible for protection. Our results indicate that SYNTGRFPPL-specific effector CTL preferentially utilize perforin-mediated cytolysis to provide protection against LP-BM5-induced pathogenesis, whereas CTL production of IFNγ is not required. Our results also suggest a minimal contribution of FasL/Fas-mediated lysis during the effector response. Collectively, these results provide insight into effector mechanisms utilized by protective CTL directed against non-traditional cryptic epitopes during disease protection. PMID:19539970

  7. Heterogeneity but individual constancy of epitopes, isotypes and avidity of factor H autoantibodies in atypical hemolytic uremic syndrome.

    PubMed

    Nozal, Pilar; Bernabéu-Herrero, Maria E; Uzonyi, Barbara; Szilágyi, Ágnes; Hyvärinen, Satu; Prohászka, Zoltán; Jokiranta, T Sakari; Sánchez-Corral, Pilar; López-Trascasa, Margarita; Józsi, Mihály

    2016-02-01

    Factor H (FH) autoantibodies are present in 6-10% of atypical hemolytic uremic syndrome (aHUS) patients, most of whom have homozygous deficiency of the FH-related protein FHR-1. Although the pathogenic role of the autoantibodies is established, little is known about their molecular characteristics and changes over time. Here, we describe the specificity and other immunological features of anti-FH autoantibodies in the Spanish and Hungarian aHUS cohorts. A total of 19 patients were included and serial samples of 14 of them were available. FH autoantibodies from FHR-1 deficient patients (n=13) mainly recognized FH, its SCR19-20 fragment and FHR-1, but autoantibody specificity in patients who are homo- or heterozygous for the CFHR1 gene (n=6) was heterogeneous. No significant changes apart from total antibody titer were observed during follow-up in each patient. Fine epitope mapping with recombinant FH SCR19-20 containing single amino acid mutations showed significantly reduced binding in 6 out of 14 patients. In most cases, autoantibody binding to residues 1183-1189 and 1210-1215 was impaired, revealing a major common autoantibody epitope. Avidities showed variations between patients, but in most cases the avidity index did not change upon time. Most autoantibodies were IgG3, and all but three presented only with kappa or with lambda light chains. Although the pathogenic role of anti-FH autoantibodies in aHUS is well established, this study shows autoantibody heterogeneity among patients, but no significant variation in their characteristics over time in each patient. The presence of a single light chain in 16 out of 19 patients and the limited number of recognized epitopes suggest a restricted autoantibody response in most patients. PMID:26703217

  8. 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

  9. Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

    PubMed Central

    Wu, Han-Chung; Yeh, Chia-Tsui; Huang, Yue-Ling; Tarn, Lih-Jeng; Lung, Chien-Cheng

    2001-01-01

    Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103 and 104 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine. PMID:11425742

  10. 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

  11. PREDICTING PREFERENTIAL ADSORPTION OF ORGANICS BY ACTIVATED CARBON

    EPA Science Inventory

    Preferential adsorption of organic compounds onto activated carbon from dilute aqueous solutions was studied to develop a comprehensive theoretical basis for predicting adsorption of multicomponent solutes. The research program investigates why some solutes are strong adsorbers, ...

  12. Emergence of global preferential attachment from local interaction

    NASA Astrophysics Data System (ADS)

    Li, Menghui; Gao, Liang; Fan, Ying; Wu, Jinshan; Di, Zengru

    2010-04-01

    Global degree/strength-based preferential attachment is widely used as an evolution mechanism of networks. But it is hard to believe that any individual can get global information and shape the network architecture based on it. In this paper, it is found that the global preferential attachment emerges from the local interaction models, including the distance-dependent preferential attachment (DDPA) evolving model of weighted networks (Li et al 2006 New J. Phys. 8 72), the acquaintance network model (Davidsen et al 2002 Phys. Rev. Lett. 88 128701) and the connecting nearest-neighbor (CNN) model (Vázquez 2003 Phys. Rev. E 67 056104). For the DDPA model and the CNN model, the attachment rate depends linearly on the degree or vertex strength, whereas for the acquaintance network model, the dependence follows a sublinear power law. It implies that for the evolution of social networks, local contact could be more fundamental than the presumed global preferential attachment.

  13. 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

  14. Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173

    PubMed Central

    Kabtni, Sarah; van den Born, Jaap; Bakker, Stephan; Navis, Gerjan; Krämer, Bernard; Yard, Benito; Hauske, Sibylle

    2016-01-01

    Background and Aims The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis. Methods and Results Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis. Conclusions Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of C