Interpreting Negative Data on Antipeptide Paratope Binding to Support Development of B-Cell Epitope Prediction for Vaccine Design and Other Translational Applications

Abstract

The design of synthetic vaccine peptides and other constructs (e.g., for developing immunodiagnostics) is informed by B-cell epitope prediction for antipeptide paratopes, which crucially depends on physicochemically and biologically meaningful interpretation of pertinent experimental data as regards paratope-epitope binding, with negative data being particularly problematic as they may be due to artefacts of immunization and immunoassays. Yet, the problem posed by negative data remains to be comprehensively addressed in a manner that clearly defines their role in the further development of B-cell epitope prediction. Hence, published negative data were surveyed and analyzed herein to identify key issues impacting on B-cell epitope prediction. Data were retrieved via searches using the Immune Epitope Database (IEDB) and review of underlying primary sources in literature to identify said issues, which include (1) inherent tendency toward false-negative data with use of solid-phase immunoassays and/or monoclonal paratopes, (2) equivocal data (i.e., both positive and negative data obtained from similar experiments), and (3) failure of antipeptide paratopes to cross-react with antigens of covalent structure and/or conformation different from that of the peptide immunogens despite apparent identity between curated epitope sequences. Analysis of experimental details thus focused on negative data from fluid-phase (e.g., immunoprecipitation) assays for detection of polyclonal paratope-epitope binding. Underlying literature references were reviewed to confirm the identification of negative data included for analysis. Furthermore, data from assays to detect cross-reaction of antipeptide antibody with protein antigen were included only if supported by positive data on either the corresponding reaction of the same antibody with peptide antigen or cross-reaction of said antibody with denatured protein antigen, to exclude the possibility that negative data on cross-reaction were due to absence of antipeptide paratopes in the first place (e.g., because of failed immunization due to insufficient immunogenicity and/or immune tolerance). Among currently available negative binding data on antipeptide antibodies, very few are on polyclonal responses yet also clearly attributable to conformational differences between peptide immunogens and native cognate proteins thereof. This dearth of negative data suitable for benchmarking B-cell epitope prediction conceivably could be addressed by generating positive data on binding of polyclonal antipeptide antibodies to cognate-protein sequences (e.g., in solid-phase immunoassays using unfolded protein antigen) to complement negative data on failure of the same antibodies to cross-react with native protein (e.g., in fluid-phase immunoassays, without artefactual covalent modification of antigens that tends to produce false-negative results). As regards cross-reactive binding of native cognate proteins by antipeptide antibodies (e.g., as mechanistic basis for novel vaccines and immunotherapeutics), negative data are most informative where attributable to conformational differences between peptide immunogens and target proteins. This is favored by careful peptide-immunogen design (e.g., avoiding covalent backbone and sidechain differences vis-a-vis target protein sequence) and positive data on antibody binding of the target protein sequence (e.g., in unfolded protein) paired with negative data on the same antibody using native protein antigen (e.g., from fluid- rather than solid-phase assays).