Development Of Brucellosis S2 Gel Live Vaccine And Evaluation Of Different Mucosal Immune Pathways

Brucellosis is a serious zoonosis caused by Brucella,Sick livestock are the main source of transmission of the disease.The main prevention and control measure of brucellosis is to immunize live brucellosis vaccine for susceptible livestock.The traditional injection of immunization has the disadvantages such as unsafe for pregnant animals and long duration of antibody which affects the differential diagnosis.After the oral live vaccine of Brucella(S2strain),it overcomes the above problems caused by injection immunization.However,oral immunization has the problem of easy loss of vaccine immunization,which poses a certain threat to the safety of operators themselves,and also causes insufficient dose of immunization due to loss.In this study,the temperature sensitive gel was used as the excipient for brucella live vaccine(S2 strain),and the phase transition from liquid state to gel state was completed in the mucosal immune site,so that the vaccine bacteria could be fixed in the immune site by gel,thus reducing the risk of vaccine outflow from the immune site.In this experiment,the star-point design method was used to synthesize 13 gel formulations,and then four alternative formulations were selected from the three-dimensional effect surface and contour map of the phase transition temperature.Finally,the physicochemical properties of the formulations were determined by microrheometer,and the optimal formulation of the blank temperature-sensitive gel was selected.The live brucellosis vaccine S2 was mixed with the blank temperature sensitive gel at a ratio of 1:9 to prepare the live brucellosis temperature sensitive gel vaccine(S2 strain).The live temperature sensitive gel vaccine was characterized and analyzed by environmental scanning electron microscopy.The following methods were used to evaluate the different mucosal immune effects of the S2live temperature sensitive gel vaccine.Firstly,the minimum immunization dose of mice immunized with the S2 live temperature sensitive gel vaccine was determined,and mice were immunized by four mucosal immunization routes(vaginal perfusion,eye,oral,rectal perfusion),to compare the amount of bacteria excreted from the body and the antibody levels of mucosal immunity and injection immunity of the S2 live temperature-sensitive gel vaccine and the conventional S2 vaccine.Secondly,the OIE recommended method was used to measure the immune effect of 4 mucosal immune pathways of S2 live temperature-sensitive gel vaccine and conventional S2 vaccine injection on mice through challenge protection test.Lastly,the sheep were immunized by the mucosal route with the better immune effect in the mouse model experiment,and the minimum immune dose of S2 live temperature-sensitive gel vaccine was determined.At th same time,the immune safety,cellular immune level and humoral immune level of different mucosal pathways(oral and vaginal)were compared.The best prescription of blank temperature sensitive gel for S2 gel live vaccine was screened by the above methods:poloxamer 407(P407):22.04%,poloxamer 188(P188):2.81%,the S2 live temperature-sensitive gel vaccine S2 was prepared by mixing with live brucellosis vaccine in 1:9.Scanning electron microscopy showed that the S2 live temperature-sensitive gel vaccine could fully carry vaccine bacteria and facilitate the release of vaccine bacteria.The minimum immune dose of the S2 live temperature-sensitive gel vaccine on mice was 100×10~8CFU,and the amount of bacteria excreted from the immune parts of mice was found to be 0?30%of that of conventional vaccine.Compared with the different mouse mucosal immunization method,the vaginal perfusion was the smallest amount of bacteria excreted from the body(less than 100 CFU),the shortest accumulating time(within 1 d),the eye instillation and rectal perfusion followed,and the oral excretion was the most of bacteria excreted from the body(1210 CFU),the results show that compared with the conventional vaccine,the S2 live temperature-sensitive gel vaccine can significantly reduce the bacteria,and the vaginal immunity has the least amount of bacteria.Comparison of antibody levels in different mucosal pathways revealed that mucosal immune antibodies were25?80%lower than injection immunization,about 45 days after imunizationthe,results of micro-tube agglutination test(MSAT)decreased to 1:50 or close to 1:50,Significantly faster than injection immunization;about 90 days after immunization,the levels of antibody in the rectum,eye,and oral administration were lower than the positive criteria.The results showed that mucosal immunity antibodies duration was shorter than injection,and mucosal immunity is helpful to differential diagnosis.The unit protection index of the immunized group(control bacterial index-immunization index)was determined by the challenge test.When the bacterial index of the control group was 6.47,the unit protection index of the immunized group was as follows:S2 conventional vaccine injection was 4.08,the S2 live temperature-sensitive gel vaccine vaginal immunity is 3.89,eye-immunization is 3.41,oral immunity is 3.32,rectal immunity is 3.25,and the results show that all four mucosal immune pathways have good protection.Among them,the immune protection effect of vaginal perfusion is more close to the traditional injection of immunization,the second is eye immunization.According to the evaluation results of the mouse model of the S2 live temperature-sensitive gel vaccine,the vaginal perfusion and eye-immunization were used to immunize the sheep.The minimum immunization dose of the S2 live temperature-sensitive gel vaccine was 500×10~8CFU.Comparing the amount of bacteria excreted from the the immune site of the sheep,it was found that the total amount(15 CFU,11 CFU)in the gel-vaccine group was lower than the conventional vaccine group(982 CFU),the results show that gel live vaccines can significantly reduce the amount of bacteria after mucosal immunization.Peripheral blood lymphocyte factor assay showed that the cytokines IFN-?,IL-2,IL-6,IL-10 and TNF-?showed significant changes after immunization and challenge,while IL-12,IL-4 and IL-8.The changes were not obvious,and the results showed that factors such as IFN-?,TNF-?,and IL-2 played an important role in the resistance to Brucella infection.The protective effect of S2 live temperature-sensitive gel vaccineon the sheep challenge test was carried out.It was found that the protection rate of the eye-immunized group and the vaginal immunization group could reach 80%or more,and the unit protection index was 2.19 and 2.42,respectively,the protection of vaginal immunity is higher.Histopathological examination revealed significant histopathological changes in the challenge control group,while the immunized group did not.The results showed that S2 live temperature-sensitive gel vaccine had a good protective effect and caused less histopathological changes.In this study,the S2 live temperature-sensitive gel vaccine was successfully prepared.The animal model of mice and sheep was used to evaluate the immune effect of different pathological methods of the gel vaccine.It was found that mucosal immunity had good immunoprotective effect and vaginal immunity was the best.It is also confirmed that the short duration of mucosal immune antibodies is advantageous for the differential diagnosis.