Articles de revues : « Epitope library »

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Moriki, Takanori, Ichiro N. Maruyama, Yusuke Yamaguchi, Atsuko Igari, Yasuo Ikeda et Mitsuru Murata. « Identification of ADAMTS13 Epitopes Required for Binding to von Willebrand Factor Using Lambda Phage Surface Display. » Blood 110, n^o 11 (16 novembre 2007) : 2707. http://dx.doi.org/10.1182/blood.v110.11.2707.2707.

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Abstract The metalloprotease ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. We planned to search core epitopes of ADAMTS13 that is required for its binding to VWF. We constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using immobilized VWF as a probe. After the first screening, the C-terminus of the spacer domain from Arg670 to Glu684 (termed as epitope-1) and the middle of the cysteine-rich domain from Arg484 to Arg507 (epitope-2) were determined as epitopes. When we added the synthetic epitope-1 peptide to the second screening, a new site, from Pro618 to Glu641 (epitope-3), was found in the middle of spacer domain. While the presence of synthetic epitope-2 peptide did not affect the subsequent screening, the presence of epitope-3 peptide enhanced the isolation of clones encoding epitope-1. These results suggest that ADAMTS13 epitopes-1, -2 and -3 may interact with each other for their binding to VWF. From screening in the presence of any combination or all of the three synthetic peptides, however, no new VWF binding site was uncovered. To examine the effect of divalent metal cations on the binding of ADAMTS13 epitopes to immobilized VWF, screening was carried out in the presence or absence of 5 mM of EDTA. No new epitope site was found. We next explored inhibitory effect of the synthetic epitope peptides on ADAMTS13 protease activity using recombinant ADAMTS13 and FRETS-VWF73 as a substrate. Synthetic epitopes-2 and -3 peptides markedly inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-1 peptide did not as efficiently as epitopes-2 and -3. The stronger inhibitory effect of epitope-3 peptide than that of epitope-1 peptide was confirmed by SDS-agarose gel electrophoresis analysis of cleavage products of denatured multimeric VWF molecules by recombinant ADAMTS13. This was consistent with the dissociation constants for the three synthetic peptides with immobilized VWF determined by surface plasmon resonance, in which epitopes-2 and -3 have higher affinities for VWF than that of epitope-1. The results described above suggest that ADAMTS13 may initially bind to immobilized VWF through the sites of epitope-1 and epitope-2 with relatively weak affinity. The binding of epitope-1 to VWF may subsequently induce the conformational change of VWF, thereby exposing a binding site for epitope-3 for the efficient catalytic cleavage of VWF by ADAMTS13.

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Ostrowski, M., J. A. Galeota, A. M. Jar, K. B. Platt, F. A. Osorio et O. J. Lopez. « Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain ». Journal of Virology 76, n^o 9 (1 mai 2002) : 4241–50. http://dx.doi.org/10.1128/jvi.76.9.4241-4250.2002.

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ABSTRACT After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

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Buchli, Rico, Rodney S. VanGundy, Sonja Plompen, Aaron D. Rennels, Nicholas J. Ede et William H. Hildebrand. « Mining for Treasures : Systematic Profiling of 8-, 9-, 10-, and 11-mer HLA-B*0702-restricted CD8+ T cell epitopes from the Influenza A/Puerto Rico/8/34 (H1N1) Virus Hemagglutinin with Potential Application in Vaccine Development (B193) ». Journal of Immunology 178, n^o 1_Supplement (1 avril 2007) : LB40. http://dx.doi.org/10.4049/jimmunol.178.supp.b193.

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Abstract Identification of T cell epitopes is of crucial importance for the development of cancer and viral T cell eliciting vaccines. Here, we define CD8+ T cell epitopes using a novel high-throughput system for the synthesis and screening of peptide libraries that include all dominant epitope lengths (8–11mers) for MHC class I molecules. These representative peptide libraries are tested for their ability to bind various MHC class I molecules in a competitive manner. This comprehensive screening strategy begins with the synthesis of an overlapping truncated peptide library containing mixes of four peptides with a common C terminus, but having a length of 8, 9, 10, or 11 residues. Following the initial screen, positive hits are resolved into individual epitope responses. In order to identify HLA-B*0702-restricted epitopes, a peptide library was prepared for the influenza A virus Hemagglutinin (HA) protein. Screening results revealed 63 high/medium affinity epitopes at various sizes. A number of these putative influenza epitopes coincide with directly discovered influenza epitopes as well as influenza epitopes reportedly recognized by CTL. In conclusion, this study demonstrates that the rapid and methodical screening of T cell immune epitope libraries can help in detecting and monitoring infections as well as enabling immune intervention strategies with respect to treatment or prevention of the disease.

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Jette, D. C., F. T. Kreutz, B. A. Malcolm, D. S. Wishart, A. A. Noujaim et M. R. Suresh. « Epitope mapping of prostate-specific antigen with monoclonal antibodies ». Clinical Chemistry 42, n^o 12 (1 décembre 1996) : 1961–69. http://dx.doi.org/10.1093/clinchem/42.12.1961.

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Abstract Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

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Chen, Longxin, Chaoyang Zhu, Hui Guo, Runting Li, Limeng Zhang, Zhenzhen Xing, Yue Song et al. « Epitope-directed antibody selection by site-specific photocrosslinking ». Science Advances 6, n^o 14 (avril 2020) : eaaz7825. http://dx.doi.org/10.1126/sciadv.aaz7825.

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Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1β and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.

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Suprun, Maria, Scott H. Sicherer, Robert A. Wood, Stacie M. Jones, Donald Y. M. Leung, A. Wesley Burks, David Dunkin et al. « Mapping Sequential IgE-Binding Epitopes on Major and Minor Egg Allergens ». International Archives of Allergy and Immunology 183, n^o 3 (24 novembre 2021) : 249–61. http://dx.doi.org/10.1159/000519618.

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Introduction: Molecular studies of hen’s egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (ses-)IgE and ses-IgG4 among baked-egg reactive or tolerant children. Methods: A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. Ses-IgE and ses-IgG4 repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared. Results: 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher ses-IgE and lower ses-IgG4 to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar ses-IgG4 but greater ses-IgE than tolerant group. A combination of OVA-sIgE with ses-IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype. Conclusion: We have created a comprehensive epitope library and showed that ses-IgE is a potential biomarker of baked-egg reactivity.

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Holzem, Achim, Jörg M. Nähring et Rainer Fischer. « Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment–pVIII fusion library ». Journal of General Virology 82, n^o 1 (1 janvier 2001) : 9–15. http://dx.doi.org/10.1099/0022-1317-82-1-9.

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This study describes the identification of the epitope recognized by the tobacco mosaic virus (TMV) coat protein (CP)-specific monoclonal antibody 29 (MAb29) by displaying a CP gene-fragment library on pVIII of filamentous phage M13. More than 80% of the clones isolated after one round of panning bound specifically to MAb29. DNA sequencing of ten randomly chosen MAb29-specific clones and subsequent sequence comparison revealed a common seven amino acid epitope (ELIRGTG) representing amino acids 131–137 of the TMV CP. The reactivity of MAb29 in competition ELISA towards glutathione S-transferase fused to this epitope was stronger than that towards full-length wild-type TMV CP, confirming the epitope sequence determined by gene-fragment phage display. This demonstrated that gene-fragment libraries displayed on the phage surface as fusion proteins with the filamentous bacteriophage gene VIII are useful tools for rapid identification of linear epitopes recognized by MAbs.

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Yuan, Tom Z., Ana G. Lujan Hernandez, Erica Keane, Qiang Liu, Fumiko Axelrod, Shweta Kailasan, Madeleine Noonan-Shueh, Mohammad Javad Aman, Aaron K. Sato et Yasmina N. Abdiche. « Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails ». Antibody Therapeutics 3, n^o 3 (juillet 2020) : 167–78. http://dx.doi.org/10.1093/abt/tbaa016.

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ABSTRACT Background Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. Methods and Results A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. Conclusions Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

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Maier, Richard H., Christina J. Maier, Raphaela Rid, Helmut Hintner, Johann W. Bauer et Kamil Önder. « Epitope Mapping of Antibodies Using a Cell Array–Based Polypeptide Library ». Journal of Biomolecular Screening 15, n^o 4 (16 mars 2010) : 418–26. http://dx.doi.org/10.1177/1087057110363821.

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The authors describe a technique for mapping the epitopes of protein antigens recognized by mono- or polyclonal antibodies. This method is based on a recombinant polypeptide library, expressed in a bacterial expression system, arrayed at high density, and tested on a membrane with automated procedures. The authors analyzed the epitope of a commercially available monoclonal antibody to vitamin D receptor (VDR). About 2300 overlapping VDR peptides were screened on a test array, and a contiguous stretch of 37 amino acids was identified as the epitope. Its authenticity was confirmed by Western blotting and an immunofluorescence competition assay on human skin tissue samples. The authors define the proposed method as a cell-based protein or peptide array that is adaptable to many applications, including epitope mapping of antibodies and autoantibodies, autoantigen detection from patient sera, whole-proteome approaches such as protein-peptide interactions, or selection of monoclonal antibodies from polyclonal sera. The advantages of this method are (a) its ease of protein array production based on well-established bacterial protein/peptide expression procedures; (b) the large number of printable colonies (as many as ~25,000) that can be arrayed per membrane; (c) there is no need for protein purification of recombinantly expressed proteins; (d) DNA, rather than protein, is the starting material to generate the arrays; and (e) its high-throughput and automatable format.

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Tsai, D. E., et J. D. Keene. « In vitro selection of RNA epitopes using autoimmune patient serum. » Journal of Immunology 150, n^o 3 (1 février 1993) : 1137–45. http://dx.doi.org/10.4049/jimmunol.150.3.1137.

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Abstract Nucleotide-specific autoimmune epitopes have not been precisely defined despite the fact that certain kinds of DNA and RNA species are known to bind autoantibodies. Our laboratory has used nucleic acid epitope libraries, consisting of randomized RNA pools, to select specific RNA conformers recognized by antibodies, including a peptide-specific antibody. In the present study, serum from a patient with systemic lupus erythematosus was used to select ligands from an RNA epitope library. The selected RNA contained sequences that were found to be similar to regions within the U1 small nuclear RNA, previously shown to react with autoantibodies. Furthermore, the selected RNA epitopes were able to inhibit autoantibody reactivity with specific regions of U1 RNA, thus demonstrating their immunologic cross-reactivity with the natural RNA epitope. Although the origins of nucleic acid-binding autoantibodies are not understood, the identification of these defined U1 RNA epitopes, in regions of the RNA where cell proteins are not known to bind, is most compatible with models of immunologic cross-reactivity or with direct presentation to the immune system rather than with anti-Id models. These experiments demonstrate that RNA epitope libraries may be used to reveal the fine specificity of autoimmune recognition and provide a useful approach to study RNA-protein interactions.

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Datta, Rohini, Rohan Roy Chowdhury, Kavyashree Manjunath, Luke Elizabeth Hanna et Raghavan Varadarajan. « A facile method of mapping HIV-1 neutralizing epitopes using chemically masked cysteines and deep sequencing ». Proceedings of the National Academy of Sciences 117, n^o 47 (9 novembre 2020) : 29584–94. http://dx.doi.org/10.1073/pnas.2010256117.

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Identification of specific epitopes targeted by neutralizing antibodies is essential to advance epitope-based vaccine design strategies. We report a facile methodology for rapid epitope mapping of neutralizing antibodies (NAbs) against HIV-1 Envelope (Env) at single-residue resolution, using Cys labeling, viral neutralization assays, and deep sequencing. This was achieved by the generation of a library of Cys mutations in Env glycoprotein on the viral surface, covalent labeling of the Cys residues using a Cys-reactive label that masks epitope residues, followed by infection of the labeled mutant virions in mammalian cells in the presence of NAbs. Env gene sequencing from NAb-resistant viruses was used to accurately delineate epitopes for the NAbs VRC01, PGT128, and PGT151. These agreed well with corresponding experimentally determined structural epitopes previously inferred from NAb:Env structures. HIV-1 infection is associated with complex and polyclonal antibody responses, typically composed of multiple antibody specificities. Deconvoluting the epitope specificities in a polyclonal response is a challenging task. We therefore extended our methodology to map multiple specificities of epitopes targeted in polyclonal sera, elicited in immunized animals as well as in an HIV-1–infected elite neutralizer capable of neutralizing tier 3 pseudoviruses with high titers. The method can be readily extended to other viruses for which convenient reverse genetics or lentiviral surface display systems are available.

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Yu, Zhiguang, et Yen-Ho Chu. « Combinatorial epitope search : Pitfalls of library design ». Bioorganic & ; Medicinal Chemistry Letters 7, n^o 1 (janvier 1997) : 95–98. http://dx.doi.org/10.1016/s0960-894x(96)00585-9.

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Blake, J., J. V. Johnston, K. E. Hellström, H. Marquardt et L. Chen. « Use of combinatorial peptide libraries to construct functional mimics of tumor epitopes recognized by MHC class I-restricted cytolytic T lymphocytes. » Journal of Experimental Medicine 184, n^o 1 (1 juillet 1996) : 121–30. http://dx.doi.org/10.1084/jem.184.1.121.

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Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.

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Henry, M., Y. Malthièry, E. Zanelli et B. Charvet. « Epitope mapping of human thyroglobulin. Heterogeneous recognition by thyroid pathologic sera. » Journal of Immunology 145, n^o 11 (1 décembre 1990) : 3692–98. http://dx.doi.org/10.4049/jimmunol.145.11.3692.

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Abstract Thyroglobulin is the major Ag of the thyroid gland involved in autoimmune pathologies. Epitope mapping was carried out with a rabbit polyclonal immune serum against fusion proteins expressed in prokaryotic cells. After screening of an initial human thyroglobulin cDNA library and subcloning of immunoreactive clones, seven epitopes were characterized and localized on the human thyroglobulin monomeric molecule. One was close to each extremity of the molecule, and five others were concentrated in the middle, covering a sixth of this 2748-amino-acid chain. The immunoreactivities of 18 autoimmune sera from different thyroid pathologies were tested against the seven previously characterized epitopes. Those from Hashimoto's thyroiditis were the most immunoreactive. Immune responses were heterogeneous for sera from different pathologies as well as for those from the same pathology. The central epitopes and the near-C-terminal epitope, however, were the epitopes most often recognized by the immune sera. These findings show that some autoepitopes overlap accurately with some heteroepitopes characterized by a polyclonal immune serum directed against the mature protein.

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Mustafa, A. S., F. Oftung, A. Deggerdal, H. K. Gill, R. A. Young et T. Godal. « Gene isolation with human T lymphocyte probes. Isolation of a gene that expresses an epitope recognized by T cells specific for Mycobacterium bovis BCG and pathogenic mycobacteria. » Journal of Immunology 141, n^o 8 (15 octobre 1988) : 2729–33. http://dx.doi.org/10.4049/jimmunol.141.8.2729.

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Abstract We have used human CD4+ T lymphocyte clones as primary probes to identify and isolate lambda gt11 rDNA clones that express epitopes recognized by T cells. The method that we describe here permits a direct survey of T cell epitope coding sequences in genomic DNA or cDNA libraries. A lambda gt11 library of Mycobacterium leprae DNA was screened with M. leprae-reactive human T cell clones as probes, allowing the isolation of a M. leprae DNA clone encoding the unidentified Ag. This DNA clone differs in restriction maps from those previously identified by antibody probes and encodes an epitope that is unique to vaccine strains of Mycobacterium bovis bacillus Calmette-Guérin and pathogenic mycobacteria. This method is generally applicable and should expedite the study of Ag and epitopes important to the T cell response in infections and in autoimmune diseases.

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Linke, Michael J., Susan M. Sunkin, Ryan P. Andrews, James R. Stringer et Peter D. Walzer. « Expression, Structure, and Location of Epitopes of the Major Surface Glycoprotein of Pneumocystis carinii f. sp. carinii ». Clinical Diagnostic Laboratory Immunology 5, n^o 1 (1 janvier 1998) : 50–57. http://dx.doi.org/10.1128/cdli.5.1.50-57.1998.

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ABSTRACT The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5′ end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein–MSG-B fusion protein (MBPMSG-B41–1065) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

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Lesénéchal, Mylène, Laurence Becquart, Xavier Lacoux, Laurent Ladavière, Renata C. P. Baida, Glaucia Paranhos-Baccalà et José Franco da Silveira. « Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections ». Clinical Diagnostic Laboratory Immunology 12, n^o 2 (février 2005) : 329–33. http://dx.doi.org/10.1128/cdli.12.2.329-333.2005.

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

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Ballmann, Rico, Sven-Kevin Hotop, Federico Bertoglio, Stephan Steinke, Philip Alexander Heine, M. Zeeshan Chaudhry, Dieter Jahn et al. « ORFeome Phage Display Reveals a Major Immunogenic Epitope on the S2 Subdomain of SARS-CoV-2 Spike Protein ». Viruses 14, n^o 6 (17 juin 2022) : 1326. http://dx.doi.org/10.3390/v14061326.

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The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.

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Scott, J., et G. Smith. « Searching for peptide ligands with an epitope library ». Science 249, n^o 4967 (27 juillet 1990) : 386–90. http://dx.doi.org/10.1126/science.1696028.

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Pedroza-Roldan, Cesar, Claudia Charles-Niño, Rafael Saavedra, Tzipe Govezensky, Luis Vaca, Eric Avaniss-Aghajani, Goar Gevorkian et Karen Manoutcharian. « Variable epitope library-based vaccines : Shooting moving targets ». Molecular Immunology 47, n^o 2-3 (décembre 2009) : 270–82. http://dx.doi.org/10.1016/j.molimm.2009.09.024.

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Rezvani, Katayoun, Stephan Mielke, Yasemin Kilical, Matthias Grube, Hiroshi Fujiwara, Giuseppe Sconocchia, Jongming Lee, Frank El-Ouriaghi, Nancy Hensel et A. John Barrett. « Identification of Novel MHC Class I and Class II Epitopes of WT1 Using a Peptide Library Screen. » Blood 106, n^o 11 (16 novembre 2005) : 2764. http://dx.doi.org/10.1182/blood.v106.11.2764.2764.

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Abstract Although several HLA-A*0201-restricted immunodominant peptides from the leukemia-restricted protein WT-1 are characterized, T cell responses to peptide sequences binding to other common class I and II epitopes of WT-1 remain almost completely unexplored. A more comprehensive definition of the WT-1 antigen would extend peptide antigen vaccines to individuals lacking HLA-A*0201 and *2402 and improve vaccine potency by recruiting both CD4+ and CD8+ T cell responses. Here we used a WT1 peptide library to identify WT-1 peptide sequences inducing CD4+ and CD8+ T cell responses in normal individuals and patients with AML and other myeloid leukemias. Six cases were studied. The library consisted of 110 15mer peptides overlapping by 11aa covering the entire WT-1 protein in 21 pools. Monocytes were isolated by plastic adherence and pulsed with peptide pools for 3 hours. Autologous CD8+ and CD4+ T cells were then added. Pools of peptides were prepared in such a way that each peptide was represented in two different peptide pools, allowing the identification of the respective peptide by responses in the two corresponding pools. Cells were harvested for RNA extraction and reverse transcription. Real time PCR (RQ-PCR) was used to identify peptide-specific induction of IFN-γ and IL-2 in CD8+ and CD4+ T cells. The SYFPEITHI binding motif software was then used to predict the probable HLA restriction for the candidate epitopes. To confirm candidate peptide immunogenecity and HLA restriction, selected peptides were synthesized and tested individually. In addition to the known HLA-A*0201 peptides WT37, WT126, WT187 and WT235 we identified 20 new MHC class I and II epitopes of WT1. Four were restricted by more than one HLA allele, demonstrating the promiscuity of epitope binding. One epitope (VPGVAPTLV) was restricted to HLA-A*0201 and HLA-B*5101. One epitope (SGQFTGTAGACRYGP) was restricted by a class I HLA allele, namely HLA-*6801 and a class II HLA allele, DR*1501. Two epitopes (YGPFGPPPPSQASGQ and QKKFARSDELVRHHN) were restricted by multiple MHC class II alleles. The proliferative response of CD4+ and CD8+ T cells to candidate peptides was confirmed using CFSE labeling. We now plan to characterize the antileukemic effects of CD4+ and CD8+ T cells induced by these peptides with a view to designing broad-spectrum vaccines inducing leukemia-reactive T cells across a wide range of HLA types.

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Ricca, George A., Victoria South, George H. Searfoss, Stephen French, Christopher Cheadle, Edward Murray, Richard Howk et Michael Jaye. « Identification of Novel Peptide Antagonists for von Willebrand Factor Binding to the Platelet Glycoprotein Ib Receptor from a Phage Epitope Library ». Thrombosis and Haemostasis 73, n^o 01 (1995) : 144–50. http://dx.doi.org/10.1055/s-0038-1653740.

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SummaryWe have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 107different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein lb interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

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Dallo, S. F., C. J. Su, J. R. Horton et J. B. Baseman. « Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence. » Journal of Experimental Medicine 167, n^o 2 (1 février 1988) : 718–23. http://dx.doi.org/10.1084/jem.167.2.718.

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A genomic library of Mycoplasma pneumoniae was constructed by cloning sheared genomic DNA into the expression vector lambda gt11. Recombinant clones were screened using anti-M. pneumoniae mAbs reactive with adhesin P1 epitopes that mediate cytadherence. 10 clones with different size inserts were isolated. These clones possessed P1 sequences localized to the COOH terminus of the P1 gene. All clones produced fusion proteins that reacted with acute and convalescent sera of patients infected with M. pneumoniae. Interestingly, one clone, P1-7, contained an epitope that was confined to a region of 13 amino acids present in the M. pneumoniae genome as a single copy. The identification of this cytadherence-related epitope permits the production of a synthetic peptide that can be used as a rational vaccine candidate and serodiagnostic probe.

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Grollo, Lara, Joseph Torresi, Heidi Drummer, Weiguang Zeng, Nicholas Williamson et David C. Jackson. « Exploiting Information Inherent in Binding Sites of Virus-Specific Antibodies : Design of An HCV Vaccine Candidate Cross-Reactive with Multiple Genotypes ». Antiviral Therapy 11, n^o 8 (novembre 2006) : 1005–14. http://dx.doi.org/10.1177/135965350601100809.

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Background/Aims The role of antibody in hepatitis C virus (HCV) infection remains unclear although many reports attest to its role in viral clearance. Here we describe epitopes that are recognized by antibody present in the serum of infected patients and show that such epitopes can induce neutralizing antibodies. Methods Human serum containing hyperimmune anti-HCV IgG was used to extract epitopes from a library of synthetic peptides that encompassed the sequences of the E1 and E2 proteins of HCV genotype 1a H77. Peptides that were bound by IgG were identified by mass spectrometry. Assembly of these epitopes with a helper T cell determinant was then carried out in order to construct candidate epitope-based vaccines. Results Three distinct antigenic sites were defined in the E1E2 glycoproteins by epitopes identified by antibody present in infected individuals. Four of the peptide epitopes identified are conserved in at least three HCV genotypes and are bound by antibody present in the sera of chronically infected and convalescent individuals. Synthetic vaccines based on these epitopes elicited antibodies that are capable of (i) capturing HCV virions from the serum of viraemic patients and (ii) inhibiting HCV pseudovirus particle entry into Huh7 cells. Conclusions This approach exploits the information inherent in the binding sites of virus-specific antibodies and represents a novel method for the design of synthetic epitope-based vaccines.

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He, Bifang, Canquan Mao, Beibei Ru, Hesong Han, Peng Zhou et Jian Huang. « Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking ». Computational and Mathematical Methods in Medicine 2013 (2013) : 1–6. http://dx.doi.org/10.1155/2013/983829.

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

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GOEDHALS, D., J. T. PAWESKA et F. J. BURT. « Identification of human linear B-cell epitope sites on the envelope glycoproteins of Crimean-Congo haemorrhagic fever virus ». Epidemiology and Infection 143, n^o 7 (4 septembre 2014) : 1451–56. http://dx.doi.org/10.1017/s0950268814002271.

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SUMMARYA peptide library was used to screen for regions containing potential linear B-cell epitope sites in the glycoproteins and nucleoprotein of Crimean-Congo haemorrhagic fever virus (CCHFV) in an enzyme-linked immunosorbent assay (ELISA). The library consisted of 156 peptides, spanning the nucleoprotein and mature GN and GC proteins in a 19-mer with 9-mer overlap format. Using pooled serum samples from convalescent patients to screen the library, six peptides were identified as potential epitope sites. Further testing of these six peptides with individual patient sera identified two of these peptides as probable epitope sites, with peptide G1451–1469 reacting to 13/15 and peptide G1613–1631 to 14/15 human sera. These peptides are situated on the GC protein at amino acid positions 1451–1469 (relative to CCHFV isolate SPU103/97) (TCTGCYACSSGISCKVRIH) and 1613–1631 (FMFGWRILFCFKCCRRTRG). Identified peptides may have application in ELISA for diagnostic or serosurveillance purposes.

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Lubahn, BC, J. Ware, DW Stafford et HM Reisner. « Identification of a F.VIII epitope recognized by a human hemophilic inhibitor ». Blood 73, n^o 2 (1 février 1989) : 497–99. http://dx.doi.org/10.1182/blood.v73.2.497.497.

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Abstract Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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Lubahn, BC, J. Ware, DW Stafford et HM Reisner. « Identification of a F.VIII epitope recognized by a human hemophilic inhibitor ». Blood 73, n^o 2 (1 février 1989) : 497–99. http://dx.doi.org/10.1182/blood.v73.2.497.bloodjournal732497.

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Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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Tsui, P., M. A. Tornetta, R. S. Ames, B. C. Bankosky, S. Griego, C. Silverman, T. Porter, G. Moore et R. W. Sweet. « Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning. » Journal of Immunology 157, n^o 2 (15 juillet 1996) : 772–80. http://dx.doi.org/10.4049/jimmunol.157.2.772.

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Abstract We isolated a large panel of human Abs directed against the respiratory syncytial virus (RSV) Ag from combinatorial phage display libraries. Following initial differentiation of the Fabs by BstNI restriction patterns, DNA sequence analysis revealed 10 different classes of VH paired with more than 35 different VL genes. All the Fabs bound with high affinity to the F Ag. However, most Fabs competed with the binding of a representative member of this group, suggesting that the Fabs recognized a common epitope on the F Ag, and none of them neutralized virus in vitro. To suppress repetitive isolation of these non-neutralizing Abs, a representative Fab was included during panning to block this common epitope on the F Ag. By this "epitope-blocked panning" approach, two novel Fabs, encoded by unique VH and VL genes, were isolated from a previously screened library. Competition binding analysis confirmed that the Fabs recognized epitopes distinct from that of the previously isolated Fabs. One of these Fabs, 516, neutralized RSV in cell culture. These activities of Fab-516 were retained upon its genetic conversion to a mAb (IgG1) and expression in mammalian cells. Our results suggest that the RSV F glycoprotein presents a dominant, non-neutralizing epitope to the human immune system, which may serve in evasion of host defenses. However, less prevalent, fusion-inhibiting Abs were revealed by blockade of this epitope during the panning process.

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Ginsburg, David, Paula L. Bockenstedt, Elizabeth A. Allen, David A. Fox, Paul A. Foster, Zaverio M. Ruggeri, Theodore S. Zimmerman et al. « Fine Mapping of Monoclonal Antibody Epitopes on Human von Willebrand Factor Using a Recombinant Peptide Library ». Thrombosis and Haemostasis 67, n^o 01 (1992) : 166–71. http://dx.doi.org/10.1055/s-0038-1648400.

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SummaryA recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in λgtll for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in λgtll as β-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand’s disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.

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Zwick, Michael B., Aran F. Labrijn, Meng Wang, Catherine Spenlehauer, Erica Ollmann Saphire, James M. Binley, John P. Moore et al. « Broadly Neutralizing Antibodies Targeted to the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1 Glycoprotein gp41 ». Journal of Virology 75, n^o 22 (15 novembre 2001) : 10892–905. http://dx.doi.org/10.1128/jvi.75.22.10892-10905.2001.

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ABSTRACT The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1MN virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

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Collar, Amanda L., Alexandria C. Linville, Susan B. Core et Kathryn M. Frietze. « Epitope-Based Vaccines against the Chlamydia trachomatis Major Outer Membrane Protein Variable Domain 4 Elicit Protection in Mice ». Vaccines 10, n^o 6 (30 mai 2022) : 875. http://dx.doi.org/10.3390/vaccines10060875.

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Chlamydia trachomatis (Ct) is the most common bacterial sexual transmitted pathogen, yet a vaccine is not currently available. Here, we used the immunogenic bacteriophage MS2 virus-like particle (VLP) technology to engineer vaccines against the Ct major outer membrane protein variable domain 4 (MOMP-VD4), which contains a conserved neutralizing epitope (TTLNPTIAG). A previously described monoclonal antibody to the MOMP-VD4 (E4 mAb) is capable of neutralizing all urogenital Ct serovars and binds this core epitope, as well as several non-contiguous amino acids. This suggests that this core epitope may require conformational context in order to elicit neutralizing antibodies to Ct. In order to identify immunogens that could elicit neutralizing antibodies to the TTLNPTIAG epitope, we used two approaches. First, we used affinity selection with a bacteriophage MS2-VLP library displaying random peptides in a constrained, surface-exposed loop to identify potential E4 mAb mimotopes. After four rounds of affinity selection, we identified a VLP-displayed peptide (HMVGSTKWTN) that could bind to the E4 mAb and elicited serum IgG that bound weakly to Ct elementary bodies by ELISA. Second, two versions of the core conserved TTLNPTIAG epitope (TTLNPTIAG and TTLNPTIAGA) were recombinantly expressed on the coat protein of the MS2 VLP in a constrained, surface-exposed loop. Mouse immune sera IgG bound to Ct elementary bodies by ELISA. Immunization with these MS2 VLPs provided protection from vaginal Chlamydia infection in a murine challenge model. These data suggest that short peptide epitopes targeting the MOMP-VD4 could be appropriate for Ct vaccine design when displayed on an immunogenic bacteriophage VLP vaccine platform.

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Jin, Young-Hee, Bongsu Kang et Byung S. Kim. « Theiler's Virus Infection Induces a Predominant Pathogenic CD4+ T Cell Response to RNA Polymerase in Susceptible SJL/J Mice ». Journal of Virology 83, n^o 21 (12 août 2009) : 10981–92. http://dx.doi.org/10.1128/jvi.01398-09.

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ABSTRACT Theiler's murine encephalomyelitis virus (TMEV)-induced immune-mediated demyelinating disease in susceptible mouse strains has been extensively investigated as a relevant model for human multiple sclerosis. Previous investigations of antiviral T-cell responses focus on immune responses to viral capsid proteins, while virtually nothing is reported on immune responses to nonstructural proteins. In this study, we have identified noncapsid regions recognized by CD4+ T cells from TMEV-infected mice using an overlapping peptide library. Interestingly, a greater number of CD4+ T cells recognizing an epitope (3D21-36) of the 3D viral RNA polymerase, in contrast to capsid epitopes, were detected in the CNS of TMEV-infected SJL mice, whereas only a minor population of CD4+ T cells from infected C57BL/6 mice recognized this region. The effects of preimmunization and tolerization with these epitopes on the development of demyelinating disease indicated that capsid-specific CD4+ T cells are protective during the early stages of viral infection, whereas 3D21-36-specific CD4+ T cells exacerbate disease development. Therefore, protective versus pathogenic CD4+ T-cell responses directed to TMEV appear to be epitope dependent, and the differences in CD4+ T-cell responses to these epitopes between susceptible and resistant mice may play an important role in the resistance or susceptibility to virally induced demyelinating disease.

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Barchan, D., M. Balass, M. C. Souroujon, E. Katchalski-Katzir et S. Fuchs. « Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library. » Journal of Immunology 155, n^o 9 (1 novembre 1995) : 4264–69. http://dx.doi.org/10.4049/jimmunol.155.9.4264.

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Abstract 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.

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Suzuki, Mina, Taiki Aoshi, Toshi Nagata et Yukio Koide. « Identification of Murine H2-Dd- and H2-Ab-Restricted T-Cell Epitopes on a Novel Protective Antigen, MPT51, of Mycobacterium tuberculosis ». Infection and Immunity 72, n^o 7 (juillet 2004) : 3829–37. http://dx.doi.org/10.1128/iai.72.7.3829-3837.2004.

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ABSTRACT Both CD4+ type 1 helper T (Th1) cells and CD8+ cytotoxic T lymphocytes (CTL) play pivotal roles in protection against Mycobacterium tuberculosis infection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-γ) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-γ. Flow cytometric analysis with intracellular IFN-γ and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with Kd, Dd, or Ld indicated that the epitope is presented by Dd. Finally, we proved that the p24-32/Dd complex is recognized by IFN-γ-producing CTL. In C57BL/6 mice, we observed H2-Ab-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine against M. tuberculosis infection.

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Ware, Jerry, John R. Toomey et Darrel W. Stafford. « Epitope Localization of Anti-Factor VIII Monoclonal Antibodies Determined by Recombinant peptides ». Thrombosis and Haemostasis 61, n^o 02 (1989) : 225–29. http://dx.doi.org/10.1055/s-0038-1646563.

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SummaryIn order to define the epitopes recognized by anti-coagulation factor VIII (FVIII) monoclonal antibodies, we have constructed a recombinant DNA epitope library from random fragments of the FVIII cDNA. The characterization of 33 different clones producing recombinant antigens in the expression vector, λgt11, has identified the epitopes for six different anti-FVIII monoclonal antibodies. The antigenic determinant for each antibody was defined by the overlapping or shared DNA sequence of multiple immunoreactive clones. One weak inhibitor of FVIII coagulant activity binds within the gly701 ser750 sequence of the FVIII sequence. An antibody which recognizes the amino-terminus of FVIII heavy-chain (within trp14 - tyr46) does not inhibit FVIII activity. Two non-neutralizing antibodies both map within residues asp807 - ser817 while two other non-neutralizing antibodies bind within lys1673 - pro1688. The general usefulness of this strategy for mapping FVIII antigenic determinants is discussed.

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Hoerner, Christian R., Michael Jhatro, Rebecca Waitz, Kathy Kamath, Minlu Zhang, Abhilash Dhal, John Shon et Alice C. Fan. « Utilizing the autoantibody immune response to tumor antigens for kidney cancer early detection. » Journal of Clinical Oncology 40, n^o 6_suppl (20 février 2022) : 369. http://dx.doi.org/10.1200/jco.2022.40.6_suppl.369.

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369 Background: Kidney cancer (renal cell carcinoma, RCC), the 8th most common U.S. cancer, is in need for better cure rates through early detection (5-year relative survival for stage I RCC: ̃95%; for stage IV RCC ̃19%). Autoantibodies are common in cancer and result from the altered expression, localization, or post-translational modification of endogenous proteins in tumor cells (autoantigens) and from the expression of mutated genes that give rise to new proteins (neoantigens). In contrast to cellular immune responses in cancer, autoantibodies are less well characterized, yet hold promise to enable cancer early detection by immune amplification of the ‘cancer signal’ while retaining specificity to cancer types including RCC. Autoantibodies may therefore be useful for kidney cancer early detection and diagnosis. Our goal was to profile the autoantibody repertoire in blood from patients with clear cell RCC (ccRCC), the most common form of RCC, in order to: 1) determine if autoantibodies can be detected in patients with early-stage and late-stage ccRCC; 2) identify common epitopes amongst ccRCC patients that could suggest common RCC antigens; and 3) determine specificity and sensitivity of potential autoantibody biomarkers for ccRCC vs. other non-cancer conditions. Methods: We use the SERA platform (https://serimmune.com/publications/) to compare putative autoantibody signal in blood from 177 patients with ccRCC, 23 with benign kidney lesions, and ̃800 healthy controls. SERA utilizes a random bacterial display 12mer peptide library of 1010 diversity in conjunction with next-generation sequencing to ascertain epitope enrichment across the entire human proteome. Results: We find significant differences in epitope repertoires in ccRCC compared to the healthy human cohort. Patients with ccRCC exhibit a rich repertoire of rare, enriched epitopes which may comprise putative autoantibody signal. This epitope signal is present with high abundance in all ccRCC stages, including stage I ccRCC. In contrast, healthy controls and patients with benign kidney lesions demonstrate more restricted repertoires. However, we do not find evidence of common ccRCC antigens: epitopes are not conserved across large subsets of ccRCC patients. Conclusions: Our initial results suggest that each patient may develop an individualized tumor-associated antibody response. Whether assessing a select epitope panel in a patient’s blood could be useful for ccRCC early detection, or even epitope diversity without needing to identify specific epitopes, warrants further study.

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Nakagawa, Terumichi, Takanori Moriki, Yusuke Yamaguchi, Atsuko Igari, Kenji Soejima, Masanori Matsumoto, Yoshihiro Fujimura et Mitsuru Murata. « Multiple Mapping of Peptide Sequences Recognized by Various Monoclonal Anti-ADAMTS13 Antibodies with Functional or Nonfunctional Effects On the Catalytic Activity. » Blood 114, n^o 22 (20 novembre 2009) : 3182. http://dx.doi.org/10.1182/blood.v114.22.3182.3182.

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Abstract Abstract 3182 Poster Board III-119 Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity. Disclosures No relevant conflicts of interest to declare.

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Hoess, R. H., A. J. Mack, H. Walton et T. M. Reilly. « Identification of a structural epitope by using a peptide library displayed on filamentous bacteriophage. » Journal of Immunology 153, n^o 2 (15 juillet 1994) : 724–29. http://dx.doi.org/10.4049/jimmunol.153.2.724.

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Abstract The screening of phage-displayed random peptide libraries has recently emerged as a powerful technique for probing Ab-Ag interactions. We have used this method to identify the epitope recognized by a mAb, CB5B10, raised against plasminogen activator inhibitor type-1 (PAI-1). Two phage libraries, displaying random hexapeptides with or without flanking cysteine residues, were screened for binding to mAb CB5B10. The selected phages were shown to contain similar peptide sequences, all of which were flanked by cysteines. When compared with the crystal structure of PAI-1, the selected peptides closely resemble the sequence of a solvent-exposed loop connecting the COOH-terminal of an alpha-helix at Phe114 to a beta-sheet at Ser119. Because of the constraints imposed by the flanking cysteine residues, the selected peptides appear to mimic the structure and the sequence of the PAI-1 epitope. Specific contacts between the amino acids displayed by the phage and the mAb were explored using site-directed mutants of the phage peptide. The effects of these substitutions on binding to the mAb correlated well with the accessibility of the corresponding residues in the PAI-1 epitope. This is the first example of the use of phage-displayed peptide libraries to identify a structural epitope.

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Liu, Hui, Yan-Li Ding, Wei Han, Mei-Yun Liu, Rui-Yang Tian, Sheng-Li Yang et Yi Gong. « Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus ». Acta Biochimica et Biophysica Sinica 36, n^o 8 (1 août 2004) : 541–47. http://dx.doi.org/10.1093/abbs/36.8.541.

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Abstract Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, Kd) of SARS-CoV E protein was 5.7×10−8 M for B10 and 8.9×10−8 M for C20. The affinity of A17 for N protein was 2.1×10−6 M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

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Sivasubramanian, Arvind, Patricia Estep, Heather Lynaugh, Yao Yu, Adam Miles, Josh Eckman, Kevin Schutz et al. « Broad epitope coverage of a human in vitro antibody library ». mAbs 9, n^o 1 (17 octobre 2016) : 29–42. http://dx.doi.org/10.1080/19420862.2016.1246096.

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Xibin, Xiao, Zhang Changqing, Zhang Ying, Zhang Ruhua, Li Jinglüe, Feng Kaitao, Sun Yun et Ye Yongzhao. « Analysis of BAC5 mcAb-related epitope using random peptide library ». Chinese-German Journal of Clinical Oncology 2, n^o 1 (mars 2003) : 39–41. http://dx.doi.org/10.1007/bf02835368.

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Ohba, Hiroyoshi, Takatoshi Soga, Takanori Tomozawa, Yoshifumi Nishikawa, Atsushi Yasuda, Asato Kojima, Takeshi Kurata et Joe Chiba. « An immunodominant neutralization epitope on the ‘thumb’ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies ». Journal of General Virology 82, n^o 4 (1 avril 2001) : 813–20. http://dx.doi.org/10.1099/0022-1317-82-4-813.

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An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the ‘helix clump’) of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.

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Leinonen, Jari, Ping Wu et Ulf-Håkan Stenman. « Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries ». Clinical Chemistry 48, n^o 12 (1 décembre 2002) : 2208–16. http://dx.doi.org/10.1093/clinchem/48.12.2208.

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Abstract Background: Prostate-specific antigen (PSA) is the most important marker for prostate cancer, but PSA concentrations determined by various assays can differ significantly because of differences in specificity of the antibodies used. To identify epitopes recognized by various monoclonal antibodies (MAbs) to PSA, we have isolated peptides that react with the paratopes of these. Methods: Six anti-PSA MAbs representing three major epitope groups were screened with five cyclic phage display peptide libraries. After selection, the peptide sequences were determined by sequencing of the relevant part of viral DNA. Binding of the phage peptides to the MAbs was monitored by immunoassay. Results: For each MAb, several paratope-binding peptides with distinct sequence motifs were identified, but only ∼10% showed similarity with the PSA sequence. Some of these correctly predicted the location of the epitopes. By sequential panning of the library with two closely related MAbs, we identified peptides reacting equally with both MAbs. When analyzed against a large panel of PSA MAbs, the peptides generally showed restricted specificity toward the MAb used for selection, but some peptides bound to several related MAbs. Conclusions: Most of the cyclic peptides selected with PSA MAbs are specific for the MAb used for selection and do not resemble any sequence on the antigen. Peptides reactive with two MAbs recognizing the same epitope can be obtained by sequential panning. This method can be used to predict the location of some epitopes, but additional methods are needed to confirm the result.

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Glamann, Joakim, Dennis R. Burton, Paul W. H. I. Parren, Henrik J. Ditzel, Karen A. Kent, Caroline Arnold, David Montefiori et Vanessa M. Hirsch. « Simian Immunodeficiency Virus (SIV) Envelope-Specific Fabs with High-Level Homologous Neutralizing Activity : Recovery from a Long-Term-Nonprogressor SIV-Infected Macaque ». Journal of Virology 72, n^o 1 (1 janvier 1998) : 585–92. http://dx.doi.org/10.1128/jvi.72.1.585-592.1998.

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ABSTRACT An antibody phage display library was constructed from RNA extracted from lymph node cells of a simian immunodeficiency virus (SIV)-infected long-term-nonprogressor macaque. Seven gp120-reactive Fabs were obtained by selection of the library against SIV monomeric gp120. Although each of the Fabs was unique in sequence, there were two distinct groups based on epitope recognition, neutralizing activity in vitro, and molecular analysis. Group 1 Fabs did not neutralize SIV and bound to a linear epitope in the V3 loop of the SIV envelope. In contrast, two of the group 2 Fabs neutralized homologous, neutralization-sensitive SIVsm isolates with high efficiency but failed to neutralize heterologous SIVmac isolates. Based on competition enzyme-linked immunosorbent assays with mouse monoclonal antibodies of known specificity, these Fabs reacted with a conformational epitope that includes domains V3 and V4 of the SIV envelope. These neutralizing and nonneutralizing Fabs provide valuable standardized and renewable reagents for studying the role of antibody in preventing or modifying SIV infection in vivo.

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Wobus, Christiane E., Barbara Hügle-Dörr, Anne Girod, Gabriele Petersen, Michael Hallek et Jürgen A. Kleinschmidt. « Monoclonal Antibodies against the Adeno-Associated Virus Type 2 (AAV-2) Capsid : Epitope Mapping and Identification of Capsid Domains Involved in AAV-2–Cell Interaction and Neutralization of AAV-2 Infection ». Journal of Virology 74, n^o 19 (1 octobre 2000) : 9281–93. http://dx.doi.org/10.1128/jvi.74.19.9281-9293.2000.

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ABSTRACT The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341–1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.

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Lochridge, Vance P., Kathryn L. Jutila, Joel W. Graff et Michele E. Hardy. « Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions ». Journal of General Virology 86, n^o 10 (1 octobre 2005) : 2799–806. http://dx.doi.org/10.1099/vir.0.81134-0.

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Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus–cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.

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Hassan, Shermarke, Guido Baselli, Kaia Palm, Frits Richard Rosendaal, Roberta Palla et Flora Peyvandi. « Factor VIII Epitope Analysis Using a Random Peptide Phage-Display Library Approach in the Sippet Cohort ». Blood 138, Supplement 1 (5 novembre 2021) : 3176. http://dx.doi.org/10.1182/blood-2021-152889.

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Abstract Background Inhibitor development is the most severe complication of hemophilia A care, and is associated with increased morbidity and mortality. Aims The aim of this study was to use a novel epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population. Methods The population consisted of 122 previously untreated patients with severe hemophilia A that were followed-up for 50 days of exposure to FVIII. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII-specific IgG epitope repertoire was assessed by means of a novel high-throughput epitope mapping technique using a random peptide phage-display library. Using this assay, a set of affinity-selected 12-mer peptide sequences (also called mimotopes) that were strongly bound by FVIII-specific antibodies were identified. These mimotopes were clustered on the basis of sequence similarity and a consensus motif was generated for each mimotope cluster. Discriminative performance of these mimotope clusters was assessed by ROC analysis. Mimotope clusters were mapped onto the 3D structure of a B-domain deleted FVIII model using a B-cell epitope prediction algorithm (Mapitope). Results The FVIII-specific antibody response is polyclonal with several mimotope clusters. The most predominant mimotope clusters in inhibitor patients were mapped to the heavy chain of the FVIII molecule. Using plasma samples taken before exposure to FVIII, three mimotopes (with the consensus motifs "QM", "PSLxWK" and "SWPHxxxxK") were identified that predicted inhibitor development (with an AUC of 0.76, 0.80 and 0.76 respectively). Conclusion Information on immunodominant epitope clusters can be used to generate novel, less immunogenic FVIII proteins and set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII. Figure 1 Figure 1. Disclosures Palm: Protobios LLC: Current Employment, Patents & Royalties: Inventor of the patent application (PCT Application No. US/14079626) filed by Protobios that covers the use of phage display method to manipulate and monitor humoral immunity.. Palla: Pfizer: Other: Travel support; Kedrion: Other: Travel support; Novonordisk: Speakers Bureau. Peyvandi: Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Sobi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

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Hiemstra, Hoebert S., Peter A. van Veelen, Nanette C. Schloot, Annemieke Geluk, Krista E. van Meijgaarden, Sabine J. M. Willemen, Jack A. M. Leunissen et al. « Definition of Natural T Cell Antigens with Mimicry Epitopes Obtained from Dedicated Synthetic Peptide Libraries ». Journal of Immunology 161, n^o 8 (15 octobre 1998) : 4078–82. http://dx.doi.org/10.4049/jimmunol.161.8.4078.

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Abstract Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

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Snewin, V. A., S. Premawansa, G. M. Kapilananda, L. Ratnayaka, P. V. Udagama, D. M. Mattei, E. Khouri et al. « Transmission blocking immunity in Plasmodium vivax malaria : antibodies raised against a peptide block parasite development in the mosquito vector. » Journal of Experimental Medicine 181, n^o 1 (1 janvier 1995) : 357–62. http://dx.doi.org/10.1084/jem.181.1.357.

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One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

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