A Contraceptive Peptide Vaccine Targeting Sulfated Glycoprotein ZP2 of the Mouse Zona Pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2 103-134 , When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2 121-140 elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2 121-140 , To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2 123-126 sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6×A/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2 k or b haplotype. In contrast, ZP2 121-140 peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2 121-140 contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2 121-140 in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2 121-140 in CFA had reduced litter sizes as compared with mice injected with CFA alone.