Abstract
The induction of immune response against gonadotrophin releasing hormone (GnRH-I) is beneficial to disrupt fertility, reduce tainting of meat and treatment of certain sex hormone dependent tumors of mammals. To achieve this, an engineered plasmid DNA vaccine coding eight repeats of GnRH-I was used in mouse model. Here the key focus was to selectively induce Th-2 response by incorporating T-helper epitopes in the vaccine construct. The vaccine was entrapped in Hemagglutinating virus of Japan envelop (HVJE), non-ionized surfactant vesicle (NISV) and Bilosome vectors to enhance immunity. Sexually mature Swiss albino female (N=32 (groups of eight) and male (N= 16, groups of four) mice were obtained from the International Center for the Diarrheal Disease Research, Bangladesh and housed in the Veterinary Teaching Hospital, Bangladesh Agricultural University, Mymensingh. Female (Group 1, 2, 3 and 4) and male (Group 5, 6, 7 and 8) mice were caged in groups and immunized with 50μg plasmid DNA vaccine/ mice in study week 0, 3, 6, 9 and 12. Group 2 and Group 6 mice were primed with vaccine in HVJE vector and boosted with phosphate buffer saline. Group 3 female and Group 7 male were immunized with NISV through subcutaneous route. Group 4 and Group 8 mice were immunized with the vaccine in Bilosome through oral route. Group 1 female and Group 5 male mice served as untreated control. The effect of immunization was studied in terms of anti-GnRH-I antibody response (OD value at A540±SD), the length and strength of estrous cycles, effect on serum testosterone level, suppression of ovarian and testicular score count, reduced fertility in vivo and expression profile of GnRH-I and its receptor mRNA in hypothalamus, pituitary and gonads. Results of indirect ELISA showed an early anti-GnRH-I IgG antibody response in Group 2 and Group 5 mice following three weeks of immunization. Group 3 and Group 6 mice showed anti GnRH-I antibody response detected in study week 6. Group 4 and Group 8 mice immunized with Bilosome showed anti GnRH-I antibody response detected in study week 9. In study week 24, the anti GnRH-I IgG response appeared higher in Group 7 (1.171±0.223) mice followed by Group 6 (1.053±0.197), Group 3 (0.982+0.231), Group 2 (0.783±0.191), Group 8 (0.591±0.193) and Group 4 (0.475±0.205) mice in contrast to no response in Group 1/ Group 5 (0.268±0.193) control. Immunization of female mice prolonged estrous cycle, which was higher in Group 3 (nine days) followed by Group 2 (eight days) and Group 4 mice (six-seven days) in compared to five days long cycle in Group 1 control. Histological evaluation of reproductive organs revealed significant reduction of ovarian and testicular score count. About 45%, 19%, 15% and 25% convoluted tubules of testis showed evidence of spermatogenesis in Group 5, 6, 7 and 8 mice respectively. There was significant reduction of serum testosterone concentration (ng/ml +S.E.M.) in Group 7 (0.63±.42, p>0.000) and Group 6 (1.58±1.27, p>0.001) mice compared to control mice (7.27±3.37). In vivo fertility assay between vaccinated and control mice showed infertility in Group 6 and Group 7 mice. Suppression of fertility (50-75%) was seen in male and female mice immunized with vaccine in Bilosome. Group 2 and Group 3 female mice showed fertility suppression in 50% cases but the number of implants was low. The prostate and uteri of the vaccinated mice were atrophied. Results of RT-PCR assay showed that the hypothalamus and gonads of immunized mice contained GnRH-I mRNA signals. Similarly the pituitary and gonads of female and male mice were also found to contain mRNA of G protein couple receptor, an indication of reversibility nature of the vaccine. This study provided evidence that plasmid DNA vaccine delivered in mice with HVJE and NISV showed higher level of anti GnRH-I antibody response and suppressed fertility. Priming of animals with plasmid DNA vaccine in HVJE and boosting in NISV could be the choice of immunization, which requires further study.