| Research objectives:The purpose of this study is to develop a novel aqueous or oil-in-water adjuvant for the Mycoplasma hyopneumoniae inactivated vaccine as well as to reduce the side effect of the vaccine. Different adjuvants were combined with the inactivated vaccine. The study would greatly help the subsequent development of the new inactivated vaccines against Mycoplasma hyopneumoniae.Test method:Study on adjuvants of Mycoplasma hyopneumoniae inactivated vaccine focused on developing a novel aqueous or oil-in-water adjuvant adjuvants.Based on previous studies, different adjuvants formulations such as carbomer974+levamisole mixture adjuvant, carbomer971+levamisole mixture adjuvant, carbomer974+levamisole+astragalus polysaccharides mixture adjuvant, carbomer974+ISCOM-matrix mixture adjuvant,chitosan+levamisole mixture adjuvant,beta-glucan+levamisole mixture adjuvant,beta-glucan+levamisole+astragalus polysaccharides mixture adjuvant,DEAE-dextran+levamisole mixture adjuvant, DEAE-dextran+levamisole+astragalus polysaccharides mixture adjuvant and other two commercial adjuvants(GEL01ST, ISA11R VG) were evaluated in present study. M. hyopneumoniae inactivated vaccines using different adjuvants were prepared and injected in mice. Specific lymphocyte proliferation and serum IgG antibodies against M. hyopneumoniae were detected to compare the effects of different adjuvants.In this study, the best immune responses of different kinds of aqueous or oil-in-water adjuvants were selected following the immune respone of the vaccinated mice models. The selected adjuvants includes:carbomer974+levamisole mixture adjuvant, carbomer974+levamisole+astragalus polysaccharides mixture adjuvant, carbomer974+ISCOM-matrix mixture adjuvant, chitosan+levamisole mixture adjuvant, and other two commercial adjuvants(GEL01ST, ISA11R VG) also evaluated in this study. M. hyopneumoniae inactivated vaccines were prepared using different adjuvants and immunized in swine. At the same time, groups of unvaccinated pigs and those vaccinated with Esso oil seepage and Pfizer inactivated vaccine were included as control, Specific lymphocyte proliferation and serum IgG antibodies against M.hyopneumoniae were detected. In addition, safety evaluation of the protective efficacy and stability of M. hyopneumoniae inactivated vaccines containing adjuvants was done by subcutaneous immunization of mice.The test results:1. The results indicated that the carbomer974+ISCOM-matrix mixture adjuvant and the chitosan+levamisole mixture adjuvant strongly induced high levels of cellular immune and humoral immune response, better than the effect of the carbomer974+levamisole mixture adjuvant, the carbomer974+levamisole+astragalus polysaccharides mixture adjuvant and the ISA11R VG adjuvant. Animals of the groups immunized beta-glucan+levamisole mixture adjuvant, DEAE-dextran+levamisole+astragalus polysaccharide mixture adjuvant and beta-glucan+levamisole+astragalus polysaccharide mixture adjuvant had obvious cellular immune response, but weak humoral immune response. Additionally, animals of the group vaccinated with GEL01ST adjuvant had obvious humoral immune response, but a weak cellular immune response.2.In the experiment of the animal, compared with the control group, carbomer974+levamisole mixture adjuvant strongly induced high levels of cellular immune and humoral immune response, better than the effect of the group of vaccinated with the inactivated vaccines,Pfizer and Esso oil seepage. GEL01ST adjuvant and ISA11R VG adjuvant had obvious humoral immune response, but weak cellular immune response, chitosan+levamisole mixture adjuvant and carbomer974+levamisole+astragalus polysaccharides mixture adjuvant had better humoral immune response although cellular immune response was not obvious. carbomer974+ISCOM-matrix mixture adjuvant failed to strongly induced high levels of cellular immune and humoral immune response.28days after vaccination, the experimemtal pigs were necropsied, to evaluate the extent of lung lesion by assigning the lesion scores and for the assessment of vaccine efficacy with the animal that received intramuscular inoculation of live vaccine. The results show that, compared with virus control, adjuvant group1(the carbomer974+levamisole), inactivated vaccine group and Esso oil seepage group had obvious protective effect(P<0.01), their protection efficiency was7/9,6/9and4/9repectively. Adjuvant group2(the carbomer974+ISCOM-matrix) and adjuvant group3(the carbomer974+levamisole+astragalus polysaccharides) had a poor protrctive efficiency and their protection efficiency only was4/9.Adjuvant group4(the chitosan+levamisole), Adjuvant group5(GEL01ST) and Adjuvant group 6(ISA11R VG) had a protective efficiency of3/9,2/8and2/8respectively, There had no significant difference against. The results demonstrate that the adjuvant group1was better and induced a better protective efficacy as compared to the rest.3. The study on the safety of vaccine containing adjuvant, there were no adverse effects.The test conclusion:1. The results of this study shows that carbomer974+ISCOM-matrix mixture adjuvant and chitosan+levamisole mixture adjuvant strongly induce high levels of cellular immune and humoral immune response, better than the effect of the carbomer974+levamisole mixture adjuvant, carbomer974+levamisole+astragalus polysaccharides mixture adjuvant and the ISA11R VG adjuvant.2. In the experiment of the animal, compared with health/virus control, the carbomer974+levamisole mixture adjuvant strongly induced high levels of cellular immune and humoral immune response, better than the effect of the group of inactivated vaccine(Pfizer) and Esso oil seepage.After infection of28d, the experimemtal pigs were necropsied, to evaluate the lesion score and the vaccine efficacy after challenge. The results show that, compared with virus control, adjuvant group1(the carbomer974+levamisole), inactivated vaccine group and Esso oil seepage group had obvious protective effect(P<0.01), their protection efficiency was7/9,6/9,4/9repectively.3. The study on the safety of vaccine containing adjuvant, there were no adverse effects.In conclution, carbomer974+levamisole mixture adjuvant not only had the best levels of cellular immune and humoral immune response, but also a higher protective effiency. This study forms the basis for the future development of effective vaccines against porcine respiratory pathogens. |
PDF Full Text Request