Effects Of Inoculation Route And Adjuvant On Immune Response Of Mice To Rabies Vaccine

Immunization route and adjuvant are important factors affecting antigen immunogenicity and immune response.In this study,the effects of inoculation route and adjuvant on the immune response of mice to rabies vaccine were evaluated.Inoculation routes involved in this study include subcutaneous(SC),intramuscular(IM),intraperitoneal(IP)and needle-free injection technology-based intradermal(ID)routes.Mice were inoculated with vaccine at same dose on weeks 0 and 1,and immune response was assessed in terms of antigen-specific antibody,antibody subtypes,neutralizing antibodies for up to 28 weeks.Live rabies virus challenge was carried out to evaluate vaccine potency.The dynamic of immune cells and inflammatory cell infiltration at the skin and muscle tissues were determined via immunohistochemistry analysis and histopathologic examination.Besides,we used Alhydrogel as positive control,and explored the effect of AS02 with different doses on the antigen-specific antibody and neutralizing antibody level induced by different dose rabies vaccine in mice.The local reaction at injection sites was also evaluated and compared between Alhydrogel and AS02.The results showed that vaccine inoculated via ID route resulted in highest antibody titer in both antigen-specific antibody and neutralizing antibody among all administration routes,while IP and IM routes are comparable in both sides,which was followed by SC route.Antibody subtype analysis showed that IP route elicited a Th1-biased immune response,while SC and IM administration caused a prominent Th2-type immune response.Unexpectedly,ID route led to a balanced Th1 and Th2 immune responses.ID route conferred a high effective protection against lethal challenge with 40 LD50 of the rabies CVS strain,which is followed by IP and IM routes.Moreover,one third dose vaccine inoculated via ID route provide comparable or higher efficacy to those of full dose vaccine with other three routes.The results of histopathology examination revealed a transient inflammatory cell infiltration at ID and IM injection sites which peaked at 48 and 24 h,respectively,after immunization.However,all reaction disappeared within one week.The results of immunohistochemistry analysis revealed more dendritic cells and Langerhans cells appeared at ID injection site than that of IM.And the number of cells in skin tissue showed a trend of increasing and subsequently decreasing,while in muscle tissue was still keep low.The result of adjuvant study showed that for the medium-dose rabies vaccine combined with high-level AS02(25ul),medium-level AS02(10ul)and positive control groups showed the highest antigen-specific antibody and neutralizing antibody in all experimental groups,which was followed by slow-level AS02(5ul)and non-adjuvant immunized groups.In case of low-dose rabies vaccine,high-level AS02(25ul)and positive control groups showed the highest antibody titer,which was followed by medium-level AS02(5ul).Low-level AS02(5ul)and the non-adjuvant immune group showed a significantly lower antibody titer than those of others.The result of skin irritation evaluation show that high-dose AS02(25ul)group elicited more irritation at injection site as evidenced by obvious erythema happened at 1h after injection.The positive and non-immune controls groups also induced weak skin irritation.However,all tissue reaction disappeared within one week.The above results suggested that AS02 can help rabies vaccine achieve improved immune response as Alhydrogel did,but with more irritation at injection sites.This study demonstrates that immunization via intradermal administration improves the efficiency of rabies vaccine,causes a transient inflammatory response at the injection site,and promotes the dynamic migration of inflammatory cells.AS02 adjuvant can enhance the immunogenicity of the vaccine,although with more irritation.This study provides important information regarding the selection of immune route and adjuvant for rabies vaccine.