Adj Uvant Effect And Mechanism Research Of Cholera Toxin A Subunit As DNA Vaccine Adjuvant

Adjuvant plays an indispensable role in vaccine design and development. Cholera toxin is one of the recognized protein vaccine adjuvants.However, its mechanism remains to be elucidated. On the basis of preliminary work, we constructed two DNA vaccines, the fusion gene vaccine of antigen encoding gene and CTA gene, and the mixture of DNA vaccine and CTA plasmid vaccine, determined their immunogenicity and explored the possible mechanisms.This research is mainly divided into two parts, one is the phenomenon of immunogenicity improved by CTA, and the other is the mechanisms. We compared HIV-1AE TRIVN, TRIVN-CTA and TRIVN+CTA on T cell immunogenicity, antibody immunogenicity, poly-functionality of specific T cells and the efficacy of the vaccines after challenge. The result showed that immune response elicited by TRIVN-CTA (1253±489SFCs/106splenocytes) and TRIVN+CTA (908±179SFCs/106splenocytes) were higher than TRIVN (642±178SFCs/106splenocytes)(p<0.05),and TRIVN-CTA was better than TRIVN+CTA. Percentage of poly-functional T cells elicited by TRIVN-CTA was higher than TRIVN and TRIVN+CTA. The higher titer of binding antibody to clade A, B and C Nef in TRIVN-CTA group showed that it was better in stimulating humoral immune response than TRIVN and TRIVN+CTA.All the three TRIVN vaccines showed protective efficacy after challenge, especially TRIVN-CTA and TRIVN+CTA which were totally protective to challenge of rTTV. All these reveal that as an adjuvant, CTA improve the immunogenicity of the vaccine especially in the fusion form.Upon these, we further explored the mechanism of CTA as an adjuvant to DNA vaccine. In vitro, we found that TRIVN-CTA and CTA can significantly improve the secretion of IL-6and IL-1β on the NIH3T3and RAW264.7cell lines respectively. Experiments on mouse showed that TRIVN-CTA improves the secretion of IL-6and IL-1β in the immune muscle tissue compared with TRIVN.All results suggested that, as a DNA vaccine adjuvant CTA can improve the secretion of IL-6and IL-1β, which may be the mechanism of immunogenicity improvement.Then we tried to find the signal pathway related to this. We found that the increased secretion of these two cytokines is closely related to the CTA’s ADP-ribosyl transferase activity (the ability of generate cAMP).In NIH3T3cells, Forskolin can greatly stimulate the secretion of IL-6, while H89(PKA inhibitor) can reduce the secretion of IL-6. In RAW cells, Forskolin can greatly stimulate the secretion of IL-1β, while H89(PKA inhibitor) can reduce the secretion of IL-1β.Next, we discussed the relationship between the CTA ADP-ribosyl transferase activity and the immunogenicity improvement. We constructed a DNA vaccine expressing OVA-mCTA, and found that its immunogenicity (48±44SFCs/106splenocytes) was weaker than OVA-CTA (98±37SFCs/106splenocytes). The results suggest that CTA improves immunogenicity by regulating the pathway of cAMP.Our results show that the immunogenicity of TRIVN-CTA is better than TRIVN and TRIVN+CTA, reflected by the poly-functionality of specific T cells, titer of antibody, and the protective efficacy after challenge. CTA can increase the secretion of IL-6and IL-1βby regulating the pathway of cAMP in local immune microenvironment. It is suggested that CTA is a promising adjuvant to DNA vaccines, and the mechanism of its function may be different when it is used in different ways.