GENERATION AND CHARACTERIZATION OF ANTIBODIES AGAINST SYNTHETIC PEPTIDES OF PORCINE ZONA-PELLUCIDA ZP3-ALPHA

Objective: Use of synthetic zona peptides as target immunogens is a promising approach to an anti-fertility vaccine. To elucidate contraceptive epitopes, we raised polyclonal antibodies against synthetic peptides of pig ZP3alpha and evaluated for immunogenicity, ability to inhibit in vitro boar sperm-pig zona attachment, and cross-reactivity with human zonae. Methods: Five synthetic peptides were chosen based on hydropathicity analysis of ZP3alpha, synthesized and coupled to keyhold limpet haemocyanin (KLH). Pairs of male rabbits were immunized with each peptide-KLH conjugate using a multi-site intradermal injection protocol. Resulting antisera were evaluated for immunoreactivity with cognate peptide and ZP3alpha/EBGD (purified endo-beta-galactosidase-digested ZP3alpha), contraceptive potential using an in vitro pig gamete bioassay, and cross-reactivity with human zonae using indirect immunofluorescence. Results: All anti-peptide-KLH sera demonstrated immunoreactivity with cognate peptide. Anti-peptide sera, except for anti-alpha-3-KLH serum, were less immunoreactive with ZP3alpha/EBGD. Antisera directed against alpha-1-KLH, alpha-2-KLH, and alpha-3-KLH demonstrated significant (P < .00001) inhibition of boar sperm-pig zona attachment. Only anti-alpha-2-KLH and anti-alpha-3-KLH sera cross-reacted with human zonae. Conclusion: Synthetic peptides of pig ZP3alpha are immunogenic, elicit antibodies cross-reactive with human zonae, and have in vitro contraceptive activity. These data support continued investigation of synthetic ZP3alpha peptides as potential target immunogens for anti-fertility vaccine development.