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A Contraceptive Peptide Vaccine Targeting Sulfated Glycoprotein ZP2 of the Mouse Zona Pellucida¹

^a Department of Pathology, University of Virginia, Charlottesville, Virginia 22908 ^b Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

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/6xA/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.

¹ Supported by NIH U54 HD29099 and RO1 AI-41236.

² Correspondence: Kenneth S.K. Tung, Departments of Pathology, University of Virginia, Old Medical School, 4th Floor, Room 4888, Charlottesville, VA 22908. FAX: 804 924 8060; kst7k{at}virginia.edu

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