Development of an auxotrophic, live-attenuated Brucella suis vaccine strain capable of expressing multimeric GnRH.

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine.

KISS1 can be used as a novel target for developing a DNA immunocastration vaccine in ram lambs.

KISS1 gene-encoding kisspeptins are critical for the onset of puberty and control of adult fertility. This study investigated whether KISS1 can be used as a novel target for immunocastration. Human KISS1 was fused with the HBsAg-S gene for constructing an antibiotic-free recombinant plasmid pKS-asd that coded for 31.168 kDa target fusion protein. Six male Hu sheep lambs were divided into two equal groups, treatment and control. The vaccine (1mg/ram lamb) prepared in saline solution was injected into lambs at weeks 0, 3 and 6 of the experiment, respectively. Vaccine efficacy was evaluated in terms of KISS1-specific IgG antibody response, serum testosterone levels, scrotal circumference, testicular weight, length and breadth, extent of testicular tissue damage, and sexual behaviour changes. The specific anti-KISS1 antibody titre in vaccinated animals was significantly higher than that in controls (p<0.05). In addition, vaccinated animals showed lower serum testosterone level, testicular weight and length and smaller scrotal circumference than those in controls (p<0.05). Spermatogenesis of seminiferous tubules in vaccinated animals was suppressed; sexual behaviours in vaccinated animals were significantly lower (p<0.05) than those in controls. In conclusion, the immunization against KISS1 in this DNA vaccine induced a strong antibody response and resulted in the suppression of gonadal function and sexual behaviour in animals, demonstrating that KISS1 can be used as a novel target for developing a DNA immunocastration vaccine.

Immunogenicity study of plasmid DNA encoding mouse cysteine-rich secretory protein-1 (mCRISP1) as a contraceptive vaccine.

PROBLEM: To examine the immunocontraceptive properties of the plasmid pcDNA-mCRISP1 and compare them to the corresponding recombinant mCRISP1 (r-mCRISP1). METHOD OF STUDY: RT-PCR and indirect immunofluorescence were performed to observe the mCRISP1 protein expression in COS-7 cells. Three groups of mice received three injections of r-mCRISP1, pcDNA-mCRISP1 or pcDNA vector, respectively. ELISA and Western blot were used to examine the immune responses and immunoreactivity of antisera. Sperm-egg penetration assay was performed to examine the effect of anti-mCRISP1 antibodies in vitro fertilization of mouse oocytes. Fertility and mean litter size were analysed by natural mating. Histological analysis was carried out to look for potential immunopathologic effects of the antibodies. RESULTS: COS-7 cells transfected with pcDNA-mCRISP1 present the expression of mCRISP1. Both r-mCRISP1 and pcDNA-mCRISP1 raised an immune response against r-mCRISP1 protein and native CRISP1 in mouse sperm. The titres of anti-mCRISP1 antibodies from DNA immunized mice were significantly lower than that of r-mCRISP1 immunized mice, but it lasted relatively longer. Male and female pcDNA-mCRISP1 injected animals presented a statistically significant reduction in their fertility with no signs of immunopathologic effects. CONCLUSION: These studies demonstrated the feasibility of generating an immune response to mCRISP1 protein by DNA vaccine and pcDNA-mCRISP1 plasmid causing significant anti-fertility potential.

Contraceptive potential of porcine and feline zona pellucida A, B and C subunits in domestic cats.

Feral cat populations are a major problem in many urban regions throughout the world, threatening biodiversity. Immunocontraception is considered as an alternative and a more humane means to control overpopulation of pest animals than current methods including trapping, poisoning and shooting. In this study, we evaluate porcine zona pellucida (ZP) polypeptide (55 kDa) and feline ZP A, B and C subunits expressed by plasmid vectors as candidate vaccines against fertility in the female domestic cat. Cats were injected subcutaneously with three doses of the ZP vaccines. Vaccinated cats were compared with naïve cats for ZP-antibody response, ovarian histology and fertility after mating. Vaccination with native porcine ZP 55 kDa polypeptide induced anti-porcine ZP antibodies detected by ELISA. However, these antibodies did not cross-react with feline ZP as assessed by immunohistochemistry and no effect on fertility in vivo was observed after mating. However, vaccination of cats with feline ZPA or feline ZPB+C DNA vectors elicited circulating antibodies specific for feline ZP as assessed by ELISA, with reactivity to native feline ZP in ovarian follicles in situ. Vaccination with feline ZPA and ZPB+C DNA did not elicit changes in ovarian histology. Although sample sizes were small, conception rates in mated females were 25 and 20% in the ZPA and ZPB+C vaccinated groups respectively, compared with 83% in the control group. We conclude that feline ZPA and ZPB+C subunits are potential candidate antigens for immunocontraceptive vaccines in the domestic cat.

CpG motif-based adjuvant as a replacement for Freund's complete adjuvant in a recombinant LHRH vaccine.

This study compared: (1) Freund's complete adjuvant and CpG oligodeoxynucleotide (ODN) 2006 in water-in-oil emulsion as adjuvants; and (2) increasing doses of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as an antigen for a contraceptive vaccine. Treatment groups (n=8 heifers/group) were: one untreated control group; five groups receiving CpG ODN with different doses of ova-LHRH (1.5; 2.3; 3.4; 5.1; and 7.6 mg); and one group receiving 3.4 mg ova-LHRH in Freund's. Heifers were immunized at weeks 0 and 14. All vaccine treatments caused gonadal regression and estrus suppression. CpG ODN is a suitable replacement for Freund's for LHRH immunization.

Molecular cloning and assessment of the immunocontraceptive potential of the zona pellucida subunit 3 from Brandt's vole (Microtus brandti).

A full-length cDNA encoding Brandt's vole (Microtus brandti) zona pellucida glycoprotein subunit 3 (vZP3) was isolated using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1254 nucleotides encoding a polypeptide of 418 amino acid residues. The deduced amino acid sequence of vZP3 revealed high overall homology with hamster (82.1%), mouse (81.3%) and rat (80.6%). A synthetic vZP3 peptide corresponding to amino acid residues 328-343 was conjugated to keyhole limpet hemocyanin (KLH-vZP3(328-343)) and used to immunise female Brandt's voles in order to test the efficacy of this peptide as a contraceptive antigen. High IgG antibody levels to the vZP3(328-343) peptide were present in the sera of female voles immunised with KLH-vZP3(328-343) and these also cross-reacted to the zona pellucida in ovaries of Brandt's vole. The fertility of the KLH-vZP3(328-343) -immunised voles was reduced by 50% compared with controls without evidence of significant ovarian pathology.

A synthetic gonadotropin-releasing hormone (GnRH) vaccine for control of fertility and hormone dependent diseases without any adjuvant.

Active immunization against self-peptides have gained widespread acceptance inspite of their low immunogenicity. Recent applications involving multiple copies of self-peptides in linear alignment and conjugation with carrier proteins appear to increase the immune response against self-peptides. As with most vaccines, however, immunogens require supplementation with adjuvants to elicit an optimum immune response. In the present study, we prepared a double-chain mini-protein with each chain containing three linear repeats of the self-peptide gonadotropin-releasing hormone (GnRH3), the hinge region of human IgG1 (hinge), and a T-helper epitope from the measles virus protein (MVP). The GnRH3-hinge-MVP mini-protein was conjugated to purified recombinant heat shock protein 65 (Hsp 65) of Mycobacterium bovis and used to immunize rats primed with subcutaneous injections of Bacillus Calmette-Guerin (BCG) in the absence of adjuvants. The GnRH3-hinge-MVP-Hsp 65 stimulated the production of specific anti-GnRH antibodies in the absence of adjuvants and the antibody titer was comparable to that produced in rats immunized with the dimeric mini-protein in the presence of Freund's adjuvant. Moreover, immunization with the adjuvant-free GnRH3-hinge-MVP-Hsp 65 induced degeneration of the reproductive organs in both male and female rats unlike those immunized in the absence of Hsp 65 or in control animals inoculated with the vehicle only. Histological examination of the affected organs showed atrophy of the seminiferous tubules with diminished spermatogenesis in the testes of male rats. In female rats, the uteri were much smaller in size and the ovaries exhibited reduced follicular development. These findings demonstrated that GnRH3-hinge-MVP-Hsp 65 mounted a strong immune response in the absence of conventional adjuvants, and could prove useful in control of fertility and the treatment of conditions/diseases where GnRH ablation is required.

[Enhancement of immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3].

To enhance the immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3, the secreted 6his-hCGbeta-C3d3 fusion protein and 6his-hCGbeta were expressed in CHO cells and purified with Ni(2+)-chelating chromatography and Sephadex G-150 column. Then we investigated the potential of three copies of C3d as the molecular adjuvant of hCGbeta protein antigen. The antibody response to hCGbeta-C3d3 conjugates was compared with those resulting from immunization with hCGbeta alone and hCGbeta plus CFA/IFA in BALB/c mice. Our results showed that the antibody titer of hCGbeta-C3d3 was 1995-fold higher than that of hCGbeta alone and the anti-serum was capable effectively neutralizing the bioactivity of hCG. The immunity-enhancing action of C3d3 was 10-fold (primer) and 32-fold (booster) greater than CFA/IFA. These results indicated that C3d3 conjugates might be a better way to overcome the poor immunogenicity of hCGbeta contraceptive vaccine.

Development of mouse-specific contraceptive vaccines: infertility in mice immunized with peptide and polyepitope antigens.

Mouse-specific immunocontraceptive peptides have been identified in mouse proteins with key roles in reproduction from sequence comparisons to other species and tested for efficacy as immunocontraceptive antigens. Peptides were derived from granulocyte-macrophage colony-stimulating factor (GMCSF), the placental 27 kDa heat-shock protein (HSP), leukemia inhibitory factor receptor (LIFR), oviduct glycoprotein (OGP), proliferin (PLF), prolactin (PRL), sperm protein SP56 and mouse zona pellucida subunits 1 and 3 (ZP1, ZP3). Fertility of female BALB/c mice was reduced after immunization with several peptides either conjugated to a carrier protein or in the form of recombinant polyepitopes. The most effective conjugated peptides (SP56, GMCSF and PRL) induced peptide-specific serum antibodies and reduced fertility by 50%. Fertility of mice was also reduced after immunization with polyepitope antigens containing up to five different peptides fused to maltose-binding protein, but antibodies were not produced against all the encoded peptides. The most effective polyepitope antigen (containing PLF, SP56, ZP1 and ZP3 peptides) reduced fertility by 50% but induced only SP56 and ZP1 antibodies. We demonstrate that lack of antibody response to a given peptide epitope (ZP3) can be overcome if repeated copies are used in the polyepitope antigen construct, but the effect varies between mouse strains. We conclude that infertility induced in mice with a range of peptide-based vaccines is dependent on antigen formulation and genetic factors but does not necessarily correlate with peptide-specific antibody levels. In light of these results, strategies to improve the efficacy of peptide-based antifertility vaccines are discussed.

Mouse-specific immunocontraceptive polyepitope vaccines.

Two mouse-specific polyepitope protein antigens comprising different combinations of sequences chosen from the mouse fertility antigens zona pellucida proteins 1 and 3 (ZP1 and ZP3), prolactin, proliferin, granulocyte-macrophage colony-stimulating factor (GM-CSF), sperm protein SP56 and T-helper cell-stimulating epitopes were produced in bacterial protein expression systems. The recombinant proteins were fused to maltose binding protein (MBP) and used to immunize female mice; their effects on fertility were assessed. Controls were immunized with either MBP only or phosphate buffered saline (PBS). One antigen construct (MBP-polyepitope B), containing mouse-specific epitopes for ZP1, ZP3, SP56 and proliferin, significantly reduced the fertility of female BALB/c mice. Fertility in this group was decreased by > 40% compared with the MBP control and the number of viable embryos was decreased by > 60%. This construct will now be used to produce the antigen in a recombinant murine cytomegalovirus for assessment as a potential mouse-specific anti-fertility vaccine for use in the control of mice in the field.