Evaluation Of The Efficacy Of Immunocastration Vaccines Composed Of GnRH Peptides With Diffrent Adjuvants

In this study,SD rats were used as a model to study the effects of various adjuvants on the compatibility of GnRH synthetic peptide castration vaccine.A set of adjuvants and antigens with good immunocastration and high safety were screened out.Provide data foundation and theoretical basis for the follow-up research and development of castration vaccine.1.Preparation of GnRH immune castration vaccineThree different antigens were prepared by solid phase peptide synthesis.The structure of G01 is Th-（GnRH）₂,and the purity is 95.52%.The structure of G01-1 is Th-（GnRH）₂-C-Palmitic,and the purity is 92.16%.The structure of G01-2 is Th-GnRH-K-（C-Palmitic,GnRH）,and the purity is 92.16%.The molecular weight agrees with the theoretical value.Choose the following eight adjuvants:（1）W/O:MONTNIDE^TMISA 61 VG;（2）W/O/W:MONTANIDE^TMISA 201 VG;（3）O/W:MONTANIDE^TMISA 15A VG;（4）water-soluble adjuvants:MONTANIDE^TMIMS 1313 VG、MONTANIDE^TMGEL 02、Alum Adjuvant、Pamica^TMand SDA7749.A total of 11 vaccines were prepared by combining the above three antigens with eight adjuvants.The stability and viscosity of the types of emulsion vaccines and the stability and turbidity of water-soluble adjuvant vaccines accord with the quality inspection standards.The sterility test was carried out according to the method of“Chinese Veterinary Pharmacopoeia（2015 Edition）”,and the results were satisfactory.The vaccines prepared can be used for subsequent experimental studies.2.Screening of emulsion adjuvants40 male SD rats with 8-week-old were randomly divided into 4 groups.The antigen is G01 and the adjuvants are MONTANIDE^TMISA 61 VG（W/O）,MONTANIDE^TMISA 201VG（W/O/W）,MONTANIDE^TMISA 15A VG.Vaccines were firstly administered at9-week-old and boosted at 11-week-old.Weighing and collecting blood every two weeks,testing serum testosterone concentration,antibody levels.All SD rats were sacrificed at23-week-old.Mouse was removed the testes and examined the size and weight and made the tissue sections.The ELISA results showed that the antibody titer of the W/O group was significantly higher than the other immune groups（P<0.01）.The serum testosterone concentration in the W/O adjuvant group and the W/O/W adjuvant group was lower than 0.1ng/m L from 6 wpv（weeks post vaccination）to the end of the experiment,which was very significantly lower than that of the O/W adjuvant group（P<0.01）.The testicular weight of the W/O adjuvant group decreased to 1/6 of the control group,and the testicular weight of the W/O/W group decreased to 1/3 of the control group.The testis tissue sections showed that the seminiferous tubule epithelial tissue in the testicular tissue of the W/O/W adjuvant group was severely atrophied,and there were only a few spermatogonial cells in the tube.The experimental results showed that the water-in-oil adjuvant and the water-in-oil-in-water adjuvant can play a good synergistic effect on the GnRH synthetic peptide castration vaccine.The serum antibody titer of the W/O group was higher,but the W/O/W adjuvant used less mineral oil and was safer.3.Screening of water-soluble adjuvants70 male SD rats with 8-week-old were randomly divided into 7 groups.The antigen is G01 and the adjuvants are MONTANIDE^TMGEL 02,Pamica^TM（2 preparation ratios）,SDA7749,MONTANIDE^TMIMS 1313 VG,Alum adjuvant.Vaccines were firstly administered at 9-week-old and boosted at 11-week-old.Weighing and collecting blood every two weeks,testing serum testosterone concentration,antibody levels.All SD rats were sacrificed at 23-week-old.Mouse was removed the testes and examined the size and weight and made the tissue sections.The ELISA results showed that the antibody titer of the GEL 02 adjuvant group was significantly higher than the other immune groups（P<0.01）.Followed by the 1313 VG group and the two Pamica^TMadjuvant groups,the peaks were comparable,but the anti-GnRH antibody titer of the Pamica^TMadjuvant group decreased rapidly.The anti-GnRH antibody titers induced by the SDA7749 group and the Alum adjuvant group were lower.The serum testosterone concentrations were less than 0.1 ng/m L from 8 wpv to the end of the experiment in the GEL 02 group.The testosterone concentrations of the other immunized groups did not fall below the effective concentration.The testicular weight of the GEL 02 adjuvant group decreased to about 1/5 of the control group.Followed by the 1313 VG adjuvant group,the testicular weight was about 1/3 of the blank control group.The testicular weight of the remaining adjuvant groups was more than 2/3 of the testicular weight of the control group.Tissue sections and Johnsen scores showed that the degree of testicular destruction in the GEL 02 group was significantly different from the rest of the immunized group（P<0.01）.Weight of SD rats in the GEL 02 group was significantly lower than the other groups from 4wpv（P<0.05）.The experimental results show that MONTANIDE^TMGEL 02 has the best synergistic effect on GnRH synthetic peptide castration vaccine,but the safety needs to be further tested.Pamica^TMadjuvant and MONTANIDE^TMIMS 1313 VG have a synergism effect on GnRH synthetic peptide castration vaccine and can be considered in combination with other carrier-active adjuvants.4.Effect of attached palmitic acid at different positions of GnRH synthetic peptide on immunocastration vaccineThis chapter used MONTANIDE^TMISA 201 VG with good immunopotentiation and safety as a candidate adjuvant.To explore the synergism effect of the addition of palmitic acid to the GnRH synthetic peptide castration vaccine at different positions in the antigen.40 male SD rats with 8-week-old were randomly divided into 4 groups.There are unmodified（G01）group,linear structure antigen（G01-1）group,branch structure antigen（G01-2）group and blank control group respectively.Vaccines were firstly administered at9-week-old and boosted at 11-week-old.All SD rats were sacrificed at 23-week-old.The blood sample will be taken every two weeks,then testing serum testosterone concentration and antibody levels.The experimental data showed that the anti-GnRH antibody titer peaks increased in the immunized group coupled with palmitic acid.The antibody titer of the branched structure group has a high peak value and a slow decline.The serum testosterone concentration of the immunized group decreased below the effective concentration（0.1 ng/m L）,and the testosterone concentration of the branched group decreased faster than the other two groups.Conclusion:The coupled of palmitic acid can effectively improve the immune effect of the GnRH synthetic peptide vaccine.The coupled of palmitic acid to the side chain has a better synergism effect on the GnRH synthetic peptide vaccine.8 adjuvants and 3 antigens were screened in this study.The MONTANIDE^TMISA201VG（W/O/W）adjuvant has good immunopotentiation and high safety.When the antigen prepared by coupling the palmitic acid to the side chain is compatible with MONTANIDE^TMISA 201VG,the immune castration effect can be further improved.It provides a data basis and theoretical basis for the development of synthetic peptide castration vaccine products.