Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

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.

Immunocontraceptive potential of the Ig-like domain of Izumo.

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.

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.

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.

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.

[Canine zona pellucida 3 (CZP3) DNA vaccination reduces mouse fertility effectively].

Objective To construct a DNA contraception vaccine targeting canine zona pellucida 3 (CZP3) antigen and assess the immunological efficacy and contraceptive effect of the vaccine. Methods The CZP3 gene was amplified from total RNA of canine ovary by reverse transcription PCR and analyzed by bioinformatics, such as ProtScale, TMHMM, Signal P, InterProScan, PREDICT PROTEIN, homology modeling, etc. The constructed DNA vaccine pcDNA-CZP3 was used to vaccinate mice, and then its immune effect and contraceptive effect were evaluated in the mice. Results The CZP3 gene had 426 amino acids with two hydrophobic regions at its N-terminal and C-terminal, respectively. The top 22 amino acids at the N-terminal of the CZP3 was the signal peptide and there was a transmembranous helix from extracellular to intracellular at the C-terminal. CZP3 also had 8 B cell epitopes. The DNA contraception vaccine pcDNA3-CZP3 induced high levels of antibody and lower average litter size of mouse compared with the blank and negative control groups significantly. Conclusion The canine contraception DNA vaccine pcDNA-CZP3 has been successfully constructed and it can reduce the mouse fertility remarkably.

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9).

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

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.

Nasal immunization using a mimovirus vaccine based on the Eppin B-cell epitope induced suppressed fertility in mice.

To elicit potent humoral immunity and produce adequate neutralizing antibody especially in the genital tract and eventually to promote its immunogenicity, we designed an Eppin B-cell-dominant-epitope-based mimovirus vaccine with an RGD motif which can be nasally inoculated into male mice. Our results indicate that this immune strategy successfully generated a high antibody response with significantly higher anti-Eppin IgA in the genital tract, and eventually achieve significant inhibition of fertility without any interference with testis function and alteration in structural integrity. The fertility rate of the females mating with the vaccinated males declined and the progeny size was greatly reduced, but the contraceptive efficacy was still far from that of immunocontraceptives for human use. However, the research showed a new contraceptive vaccine construction and inoculation avenue, that is, mimovirus vaccine delivered nasally. Further investigation geared toward improving fertility inhibition efficacy using this inoculation strategy still remains to be explored.

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and ß-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 µg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive 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.

DNA immunization against proacrosin impairs fertility in male mice.

PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.

Phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife.

Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species' reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs.

[A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

AIM: To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway. METHODS: Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level. The female C57BL/6 mice were immunized by three ways: intramuscular injection, intranasal or oral route.Antibody levels and types were detected by ELISA. RESULTS: ELISA results showed that recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 immunization induced specific IgG, IgA levels were significantly different comparing with control (P<0.01). Antifertility experiment showed that the experimental group reduced the average fertility significantly different compared with the control group (P<0.01). CONCLUSION: Restriction analysis, RT-PCR and Western blot showed that the recombinant plasmid constructed correctly and can be the expression of mRNA and protein levels.It resulted that the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 can induce the specific immune response efficiently and enhance the immunocontraceptive effects.

Screening and identification of dominant functional fragments of human epididymal protease inhibitor.

To lay a foundation for the further development of a novel contraceptive vaccine based on epididymal protease inhibitor (Eppin) protein, possible B-cell epitopes were predicted using bioinformatics. Overlapping KLH-coupled peptides corresponding to the 89-133 amino acid domain of Eppin protein were synthesized by the Fmoc method. The male mice were immunized with various peptides resulting in a gradual elevation of specific serum IgG antibodies until they peaked at the seventh week. Treatment with antiserum in immunized mice caused a significant decrease in the sperm-egg binding rate. Eventually, antifertility assays in vivo showed that, the pregnancy rate and the number of births per labor in experimental mice were significantly decreased. The decrease in the F5 and the F4 groups were more outstanding therefore these peptides might be the dominant functional fragments of Eppin protein and provide an experimental basis for the development of effective contraceptive vaccine based on Eppin protein.

Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector.

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Immunogenicity of zona pellucida glycoprotein-3 and spermatozoa YLP(12) peptides presented on Johnson grass mosaic virus-like particles.

For safer and effective immunocontraception, zona (ZP3) and spermatozoa specific (YLP(12)) peptides have been presented on virus-like particles (VLPs) derived from Johnson grass mosaic virus coat protein. Immunization of FvB/cJ female mice with VLPs presenting YLP(12)-ZP3 fusion peptide and a physical mixture of VLPs presenting either YLP(12) or ZP3 epitope led to generation of specific antibody responses and a significant reduction in litters born per mice (p<0.005). Significant curtailment of fertility was also observed in animals immunized with adjuvnated ZP3 and YLP(12) synthetic peptides. These results suggest that VLPs can be used to present gamete epitopes for immunocontraception.

In vitro and in vivo studies evaluating recombinant plasmid pCXN2-mIzumo as a potential immunocontraceptive antigen.

PROBLEMS: Study on feasibility of pCXN2-mIzumo as a potential immunocontraceptive antigen. METHOD OF STUDY: Two groups of mice received 100 microg/mouse plasmids of pCXN2-mIzumo and pCXN2 respectively. RT-PCR Immunofluorescence assay and ELISA were performed to observe pCXN2-mIzumo expression and antibody response in the inoculated mice. Sperm penetration assay and animal mating were employed to detect differences of in vitro fertilization (IVF) rate and mean litter size between the experimental and control groups. RESULTS: Izumo cDNA positive bands were detected in sample from mice immunized with pCXN2-mIzumo. IgG response started to rise at 2 weeks after first boost and reached the highest antibody titers at 2 weeks after third boost of immunization with pCXN2-mIzumo in the experimental mice. In vitro fertilization rate in the experimental group (11.57%) was significantly lower than that in control (36.60%). Significant difference of mean litter size between female experimental and control groups was observed, and there was significant negative correlation between individual anti-serum titers and litter size (r = -0.308, P < 0.05). CONCLUSION: pCXN2-mIzumo plasmid possesses appreciable anti-fertility potential.

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.