Comparison of pig and rabbit zonae pellucidae vaccination in the female baboon (papio anuhis): analysis of immunogenicity and ovarian pathology

Abstract

Immunocontraception using zona pellucida (ZP) antigens has been considered to have great potential. However, the variations in the immune response to ZP proteins in different species is great. Since little is known about the immune responses to ZP of different species in non-human primates, an experimental study was conducted to compare the immunogenicity of native rabbit ZP and native pig ZP in the female baboon (Papio anubis) and to evaluate the effect of immunization on ovarian function. Eleven cycling female baboons were randomly divided into three groups comprising: (1) three controls immunized with TitreMax® adjuvant alone; (2) four females immunized with 450 Ilg each of pig ZP in TitreMax® adjuvant and; (3) four females immunized with 450 ug each of rabbit ZP in TitreMax® adjuvant. Each animal was immunized three times (150 ug immunogen at each immunization) at 21-day intervals. Blood, collected at the time of each immunization and monthly thereafter, was examined for the presence of antibodies against rabbit, pig and baboon ZP. Unilateral ovariectomies were initiated at 50 days after the primary immunization, and the ovaries subjected to histological analysis. Ovarian cyclicity was monitored by daily scoring of sex skin swelling. ELISA results showed that antibodies were produced against the respective immunogen (native rabbit or pig ZP) with titres being higher for the rabbit ZP group. The ELISA results also showed that sera from the rabbit ZP-immunized group recognized native pig ZP and sera from the pig ZP-immunized group recognized 111 native rabbit ZP. In addition to verifying the ELISA results, immunoblot analysis also revealed that the antisera recognized bacterially produced recombinant (BPR) rabbit ZP antigen. Interestingly, sera from all pig ZP-immunized animals only recognized BPR protein encoding the 75a portion of the 75-kDa rabbit ZP protein, while sera from each animal immunized with rabbit ZP recognized all the recombinant rabbit ZP proteins tested (55-kDa and 75a and 75b portions of the 75-kDa protein). Immunohistochemistry on sections of normal baboon ovary probed with immune baboon sera against rabbit or pig ZP demonstrated the induction of auto-antibodies against baboon ZP. Histological analysis of ovaries removed from immunized animals did not indicate any overt pathology, and all animals continued to cycle normally, as evidenced by scoring of sex skin swelling, for the 12 months of the study. Collectively, these results show that heteroimmunization of female baboons with either native rabbit ZP or native pig ZP in TitreMax® adjuvant results in the production of antibody populations that not only recognize their respective immunogen and native baboon ZP but also BPR protein encoding portions of the rabbit ZP antigen. In addition, these antibody populations recognize the cross-reactive native ZP (that is pig ZP for the rabbit ZP-immunized group and vice versa). None of these antibody populations result in any overt ovarian pathology. It can therefore be concluded that there is a difference III terms of immunogenicity and none in terms of ovarian pathology between immunization with native pig ZP and native rabbit ZP using TitreMax® adjuvant in female baboons. However, it would also be important to evaluate the effects of these antibody populations on sperm-egg binding and in vivo fertility.