Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes...

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.