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.
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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. (+info)
Use of a murine cytomegalovirus K181-derived bacterial artificial chromosome as a vaccine vector for immunocontraception.
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Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181(Perth) strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors. (+info)
Induction of castration by immunization of male dogs with recombinant gonadotropin-releasing hormone (GnRH)-canine distemper virus (CDV) T helper cell epitope p35.
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Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets. (+info)
Transmission of two Australian strains of murine cytomegalovirus (MCMV) in enclosure populations of house mice (Mus domesticus).
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To control plagues of free-living mice (Mus domesticus) in Australia, a recombinant murine cytomegalovirus (MCMV) expressing fertility proteins is being developed as an immunocontraceptive agent. Real-time quantitative PCR was used to monitor the transmission of two genetically variable field strains of MCMV through mouse populations after 25% of founding mice were infected with the N1 strain, followed by the G4 strain 6 weeks later. Pathogen-free wild-derived mice were released into outdoor enclosures located in northwestern Victoria (Australia). Of those mice not originally inoculated with virus, N1 DNA was detected in more than 80% of founder mice and a third of their offspring and similarly, G4 DNA was detected in 13% of founder mice and in 3% of their offspring. Thus, prior immunity to N1 did not prevent transmission of G4. This result is promising for successful transmission of an immunocontraceptive vaccine through Australian mouse populations where MCMV infection is endemic. (+info)
Recent advances in contraceptive vaccine development: a mini-review.
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Contraceptive vaccines (CV) may provide viable and valuable alternatives to the presently available methods of contraception. The molecules that are being explored for CV development either target gamete production [luteinizing hormone-releasing hormone (LHRH)/GnRH, FSH], gamete function [sperm antigens and oocyte zona pellucida (ZP)], and gamete outcome (HCG). CV targeting gamete production have shown varied degrees of efficacy; however, they either affect sex steroids causing impotency and/or show only a partial rather than a complete effect in inhibiting gametogenesis. However, vaccines based on LHRH/GnRH are being developed by several pharmaceutical companies as substitutes for castration of domestic pets, farm and wild animals, and for therapeutic anticancer purposes such as in prostatic hypertrophy and carcinoma. These vaccines may also find applications in clinical situations that require the inhibition of increased secretions of sex steroids, such as in uterine fibroids, polycystic ovary syndrome, endometriosis and precocious puberty. CV targeting molecules involved in gamete function such as sperm antigens and ZP proteins are exciting choices. Sperm constitute the most promising and exciting target for CV. Several sperm-specific antigens have been delineated in several laboratories and are being actively explored for CV development. Studies are focused on delineating appropriate sperm-specific epitopes, and increasing the immunogenicity (specifically in the local genital tract) and efficacy on the vaccines. Anti-sperm antibody (ASA)-mediated immunoinfertility provides a naturally occurring model to indicate how a vaccine might work in humans. Vaccines based on ZP proteins are quite efficacious in producing contraceptive effects, but may induce oophoritis, affecting sex steroids. They are being successfully tested to control feral populations of dogs, deer, horses and elephants, and populations of several species of zoo animals. The current research for human applicability is focused on delineating infertility-related epitopes (B-cell epitopes) from oophoritis-inducing epitopes (T-cell epitopes). Vaccines targeting gamete outcome primarily focus on the HCG molecule. The HCG vaccine is the first vaccine to undergo Phase I and II clinical trials in humans. Both efficacy and lack of immunopathology have been reasonably well demonstrated for this vaccine. At the present time, studies are focused on increasing the immunogenicity and efficacy of the birth control vaccine, and examining its clinical applications in various HCG-producing cancers. The present article will focus on the current status of the anti-sperm, anti-ZP, anti-LHRH/GnRH and anti-HCG vaccines. (+info)
Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3.
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Recombinant myxoma viruses expressing rabbit zona pellucida 2 (rZP2) or rabbit zona pellucida 3 (rZP3) glycoproteins were constructed and tested in domestic rabbits to assess their potential to induce autoimmune infertility. The recombinant virus expressing rZP2 had no effect on fertility or ovarian histology, despite all animals developing antibodies against the rZP2 antigen. However, recombinant viruses expressing rZP3 induced infertility in 70% of animals at the first breeding. Serum antibodies were relatively short-lived, but antibody was bound to zona pellucida of all rabbits from Day 10 onward. There was no obvious correlation between infertility and rZP3 antibody titer. There was a transient inflammatory response in the ovaries of rZP3-immunized rabbits at Day 15 but no T-cell response to rZP3 could be detected at any time. Dysfunctional follicular formation was present in ovaries from rabbits infected with rZP3-expressing viruses 15-40 days postinfection but this had disappeared at later time points. A recombinant myxoma virus expressing a modified rZP3 antigen with the C-terminal hydrophobic putative anchor sequence deleted was also tested. This virus did not induce either infertility or an antibody response against the zona pellucida. Thus, the context of antigen presentation was crucial for an autoimmune response. (+info)
Study on the antifertility effects of the plasmid DNA vaccine expressing partial brLDH-C4'.
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Partial cDNA sequence coding for Microtus brandti radde (Brandt's vole) testes-specific lactate dehydrogenase (brLDH-C4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR). By inserting the product into the eukaryotic expression vector pCR3.1, pCR3.1-brLDH-C4' was obtained as the prototype of contraceptive DNA vaccine. Immunization with pCR3.1-brLDH-C4' in BALB/c mice generated antibodies specific to purified brLDH-C4' and native mouse LDH-C4 protein. The birth rate of the pCR3.1-brLDH-C4' immunized mice was found to be decreased significantly (80% lower than that of those immunized with pCR3.1). Functions of the elicited antibodies in sera from pCR3.1-brLDH-C4' inoculated mice were further explored. The results indicated that the antibodies from the mice injected with pCR3.1-brLDH-C4' could cause the agglutination of normal sperm suspension, while the ovarian structure and the development of ovarian follicles of these mice were not impaired, which gives a possible explanation for the immunocontraceptive effects of the pCR3.1-brLDH-C4' DNA vaccine. (+info)
The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate.
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The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents. (+info)