Immunocontraception in mice using repeated, multi-antigen peptides: immunization with purified recombinant antigens.

Two immunocontraceptive antigens (AgE and AgF) were constructed that included different combinations of highly species-specific peptides from the mouse reproductive antigens SP56, ZP3, ZP2, and ZP1 in the form of multi-antigen peptides (MAPs). Both AgE and AgF contained three tandem repeats each of ZP2 and ZP3 peptide epitopes and a single copy of a ZP1 peptide sequence all of which had previously been demonstrated to individually have immunodominant or contraceptive effects. In addition, AgF contained a single contraceptive peptide derived from SP56, the putative ZP3 receptor protein on sperm. The antigens were expressed and affinity purified as recombinant repeated multi-antigen (polyepitope) peptides using an Escherichia coli maltose binding protein (MBP) expression system. Female BALB/c mice actively immunized with these antigens in Freund's adjuvants produced variable serum antibody responses to the component peptides. Fertility rates for animals immunized with AgE (40%) and AgF (20%) were significantly reduced compared to MBP immunized mice (90%), but the reduction in fertility did not correlate with peptide-specific serum antibody levels. Ovaries from all immunized mice appeared histologically normal with no evidence of oophoritis. These results demonstrate that high levels of immunocontraception can be achieved in mice, without apparent side-effects, using species-specific immunogens that include repeated peptides from proteins involved in fertilization.

Experimental inoculation of European red foxes with recombinant vaccinia virus expressing zona pellucida C proteins.

The antifertility potential of zona pellucida proteins was tested in European red foxes by immunizing females with recombinant vaccinia viruses that express zona pellucida subunit C proteins. The fox zona pellucida C (fZPC) protein was newly identified and isolated as a cDNA clone from fox ovary RNA. Eighteen European foxes were inoculated with the recombinant vaccinia viruses or with wildtype vaccinia virus (wtVV) and their clinical, virological and immune responses evaluated. Following intradermal inoculation with wtVV or recombinant vaccinia virus expressing fox zona pellucida C (rVV-fZPC), or after peroral administration with recombinant vaccinia virus expressing the porcine zona pellucida C protein (rVV-pZPC) clinical signs of disease were not observed. Five out of six foxes developed antibodies to wtVV proteins. However, none of 12 foxes (six inoculated intradermally with rVV-fZPC, six perorally with rVV-pZPC) reacted in immunoblots with the transgenic fZPC or pZPC, respectively. Infectious wtVV, rVV-fZPC or rVV-pZPC was not isolated from mucosal secretions of any of the foxes whereas viral DNA was present in oral swabs of 3/18 foxes as determined by PCR. Hematological parameters remained mostly unchanged. Histopathological changes were not observed in the ovaries of rVV-fZPC or wtVV inoculated foxes. The data indicate that inoculation of foxes with cell culture infectious wtVV, rVV-fZPC or rVV-pZPC did not result in production of infectious progeny virus in vivo. For this reason transgene expression may have been insufficient to induce adequate immune responses against the transgenic proteins.

The efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4.

Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20 mg/kg or a single dose of 100 mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-gamma expression, suggestive of an alternative or highly localized suppressive mechanism.

Contraception in mice immunized with recombinant zona pellucida subunit 3 proteins correlates with Th2 responses and the levels of interleukin 4 expressed by CD4+ cells.

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 CD4+ 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.