Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1).

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.

Immunogenicity of zona pellucida vaccines.

Development of zona pellucida (ZP) based contraceptive vaccines raises a number of complications that challenge current immunological capabilities. Our research examines two aspects of these immunological problems. First, recent studies demonstrate that one bacterially expressed rabbit ZP recombinant vaccine (rec55) induces autoantibodies in primates that prevent sperm-ZP binding and induction of the acrosome reaction in the homologous ZP in vitro. Immunization with rec55 does not induce ovarian pathology, and the duration of antibody titres indicates that this vaccine would have reversible effects on fertility. However, the immunogenicity of the rec55 protein produced in the pEX bacterial expression vector is low. We have therefore expressed the cDNA encoding rec55 using the pGEX vector for the following reasons: (i) the pGEX expressed recombinant proteins are soluble in aqueous solution; (ii) affinity purification of recombinant proteins on glutathione Sepharose columns is more effective for obtaining larger quantities of purified protein; and (iii) the availability of protease cleavage sites between the ZP and glutathione S transferase fusion proteins should eliminate the possibility of carrier-mediated suppression of immune responses. These improvements in protein production and purification yield immunogen which is more malleable to immunological studies. Second, while it is clear that ZP recombinant vaccines can eliminate the problem of ovarian pathology, it is important to understand how such pathology results from immunization with native ZP proteins and certain recombinant ZP proteins. To this end, we have initiated immunization studies in baboons comparing rabbit ZP and pig ZP immunogens in Titremax adjuvant. Unilateral ovariectomies at different time points after immunization (1.5, 2, 4 and 6 months) will allow studies on the time course of pathology within the ovary. Comparison of these results with ongoing rabbit ZP immunizations will help elucidate the differences in immunogenicity of ZP proteins isolated from the two species.