Studies On Enhancing Immune Effects Of Adjuvants To Influenza HA DNA Vaccine

The objective of this study was to find effective cytokine adjuvants for influenza virus HA DNA vaccine. To achieve this, I obtained cytokine genes including CD40L, CD28, IL-12, IL-6, and IL-8 from mouse splenocytes cDNA library and cloned them respectively into expression vector. The adjuvant effect on influenza A/PR8/34 virus HA DNA was tested by coimmunization of BALB/c mice with HA DNA plus adjuvant DNA. Each DNA at a dose of 10ug or 30ug was administered twice in a 3-week interval. One week after the second vaccination, mice were challenged with a lethal dose of PR8 virus. The effectiveness of adjuvant was evaluated by increased anti-HA antibody response, survival rate and decreased loss of body weight in mice compared with those of immunization with HA DNA alone. In addition, I also constructed expression plasmids including CD40L-HA, 2CD40L-HA, and 3CD40L-HA as fusion-type DNA vaccines and analysed their ability to protect mice against virus challenge. The results were as follows:(1) antibody responses:After the first immunization and the second immunization, the IgG antibody were detected by ELISA. The result showed that, when CD40L, IL-12 or IL-8 was coinjected with HA could elicited a higher level of specific IgG against HA than in control groups(HA alone or sham injection group) (p<0.05) ; While existence of CD28 or IL-6 had no or little effect on enhancing anti-HA antibody response (p<0.05) ; The fusion plasmids of HA and CD40L decreased antibody response. It suggests that CD40L, IL-12 and IL-8 can effectively enhance homoral immunity of BALB/c mice induced by HA DNA.(2) IgG subclasse anaylsis:After the second immunization, the coadministrated group (CD40Lplus HA, IL-12 plus HA or IL-8 plus HA ) showed only slightly increased titer of HA-speciflc IgGl Ab but pronounced abundance of IgG2a Ab compared with the HA DNA-administrated group and control group (p<0.05) , suggesting that the expressing of CD40L, IL-12 and IL-8 preferentially arguments the Th1-dependent response, as reflected by the enhancement of IgG2a Ab production.(3) Protection induced by HA DNA plus adjuvant DNA against influenza virus infection.One week after the second immunization, mice were challenged with a lethal dose ( 40 LD50 ) of PR8 virus. Body weight on postinfection and survival rate of mice after challenge were measured to evaluate the adjuvant effects of CD40L, CD28, IL-8, IL-6 and IL-12 DNA on HA DNA vaccine and HA fuse CD40L in protecting mice against acute lung infection. The results showed that co-injection of CD40L plus HA DNA gave mice lower weight loss than injection of HA DNA alone. Coinjection of IL-12 or IL-8 plus HA DNA not only gave mice lower weight loss but also enhanced survival rate; coinjection of CD28 or IL-6 plus HA had no effect in protecting mice from influenza virus challenge than that of HA alone, however HA fuse CD40L had overall detrimental effects on protection status.