The immune responses and contraceptive effect in mice were tested following immunization with purified recombinant zona pellucida (ZP) proteins produced using a vaccinia (v) virus T7 mammalian expression system. Female BALB/c and CBA mice were immunized with recombinant mouse (m) ZP3 (vmZP3) or pig (p) ZPC (vpZPC) using Freund's adjuvants and boosted three times. Fertility and mean litter size were significantly reduced in groups of BALB/c mice immunized with recombinant vmZP3 and vpZPC compared with controls treated with Freund's adjuvants alone. In CBA mice, fertility and mean litter size were significantly reduced in groups of animals immunized with vmZP3 but not with vpZPC compared with the controls. Most infertile animals treated with vmZP3 and a single infertile BALB/c mouse treated with vpZPC lacked mature follicles in the ovaries, whilst no abnormalities were detected in the remaining vpZPC treated, fertile vmZP3 treated and control mice. All mice (both fertile and infertile) immunized with vmZP3 and vpZPC produced IgG antibodies, but the levels of total IgG, IgG1 and IgG2a did not correlate with infertility. All BALB/c and CBA mice immunized with vmZP3 and vpZPC showed greater delayed type hypersensitivity responses in the footpads after challenge with their respective antigens than controls, but these did not differ between the fertile and infertile mice. There was, however, a significant correlation between infertility and the levels of the Type 2 T helper cell (Th2) cytokine interleukin 4 produced by CD41 cells from vmZP3 immunized mice in response to stimulation with vmZP3 and this did not apply to the levels of the Type 1 T helper cell (Th1) cytokine interferon gamma or the general proliferation response. The results support the conclusion that induction of Th2 responses in individual mice determines whether infertility develops in response to immunization with zona pellucida proteins.

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Mouse zona pellucida subunit 3 (mZP3) was tested for efficacy as an immunocontraceptive antigen by comparing the fertility of mice immunized with recombinant mZP3 proteins. Recombinant protein was expressed using either the vaccinia virus T7 mammalian (vmZP3 protein) or baculovirus insect cell (bmZP3 protein)-expression systems. Female BALB/c or wild mice were immunized by i.p. injection using Freund's complete adjuvant and boosted three times with affinity purified recombinant proteins in Freund's incomplete adjuvant. Most mice developed antibodies that crossreacted to the respective mZP3 antigens by ELISA or western blot. In BALB/c mice immunized with vmZP3, fertility and mean litter size were reduced transiently to 25% and 10%, respectively, of those of control mice. However, immunization with bmZP3 did not affect either the fertility or mean litter sizes in BALB/c or wild mice immunized with bmZP3. The results demonstrate that reduction in fertility can be achieved in female BALB/c mice immunized using Freund's adjuvants and recombinant mZP3 protein produced in a mammalian, but not an insect, cell-expression system. Arguments are presented for the likely role of glycosylation of the mZP3 antigen in inducing contraceptive immune responses.

Mouse PH20 (mPH20), the mouse homologue to guinea pig hyaluronidase protein PH20 (gpPH20), was used to produce contraceptive vaccines that target both sexes of mice. Previously, immunization with a female gamete antigen (the zona pellucida subunit 3 protein) delivered in a recombinant murine cytomegalovirus (MCMV), or as a purified recombinant protein, has been shown to induce infertility in female mice. There is evidence, however, that sperm protein antigens could provide broader contraceptive coverage by affecting both males and females, and the most promising has been gpPH20 when tested in a guinea pig model. Mice were therefore either inoculated with a recombinant MCMV expressing mPH20 or immunized directly with purified recombinant mPH20 protein fused to maltose-binding protein. Mice treated with either vaccine formulation developed serum antibodies that cross-reacted to a protein band of 55 kDa corresponding to mPH20 in Western blots of mouse sperm. However, there was no significant reduction in the fertility of males or females compared with control animals with either formulation. We conclude from our data that recombinant mPH20 is not a useful antigen for inclusion in immunocontraceptive vaccines that target mice.

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.