Exploration Of Using CTA/CTB As Adjuvant For HIV-1 Regulatory/accessory Gene DNA Vaccine

Human Immunodeficiency Virus Type I (HIV-1) is the etiologic agent for acquired immunodeficiency syndrome (AIDS) and there are 33.4 million people reported living with HIV/AIDS world widely (the prevalence rate is 6‰approximately), including around 700,000 cases from China. Once HIV-1 infection occurs, it is hardly to be eradicated by host immunity. Epidemiological data showed that several subtypes and CRFs of HIV-1 circulate in China, CRF07_BC, CRF08_BC, B'(Thailand B) and CRF01_AE are the major forms.Currently, sexual contact has become the dominant risk factor of HIV infection in China.CRF01_AE usually spread with sexual transmitted population is on the rise consequently. Therefore, our lab has developed a prophylactic vaccine against CRF_AE, which is desirable for controlling HIV-1 pandemic in China. However,The AE-TRIVN DNA vaccine has been proven to be a poor immunogen, far less than the specific T cell responses activated by HIV-1 structure protein,such as Env. Here, in order to improve the immunogenicity of this DNA vaccine, in this study, we used cholera toxin (Cholera enterotoxin, CT), A subunit (CTA) and the B subunit (CTB) as adjuvants and determined their adjuvant effects. Mice were immunized with 100μg of DNA vaccine either mixed with CT and its subunit or fused to direct the coincident expression of CTA or CTB.Cholera toxin (CT) is a cholera-secreted, heat-labile enterotoxin with potent adjuvant and immunomodulatory activities, it has been well studied as a mucosal immunity adjuvant. In this study, we provided another more convenient approach to express recombinant CTA or CTB in E.coli than ever before. We cloned wild type CTA or CTB gene into a commercially accessible prokaryotic expression vector pET-30a (Invitrogen) and we found that when a low final concentration of chloramphenicol was added, the expressing efficiency of CTB could be elevated to a relatively high level.The recombinant protein expressed in our system could be purified with less labor-consuming by using the method of Ni2+ affinity chromatography and it could bind with GM1 at high affinity.To determine the immune responses elicited by DNA vaccine mixed together with either CTA or CTB, we immunized mice four times at 2-week interval, and immune responses were examined in 2 weeks after the final injection. ELISPOT based on IFN-γsecreting showed that the specific T cell responses elicited by pSV1.0-TRIVN (520±150 SFCs/106 splenocytes), which was lower than the regimen together with CTB (734±240 SFCs/106 splenocytes), CTA regimen(692±220 SFCs/106 splenocytes). Although did not show significant differences, the total responses against peptide pools showed a slightly improvement.For further improving the immugenity of the DNA vaccine,we co-expressed TRIVN-CTA,TRIVN-CTB with overlap pcr. ELISPOT based on IFN-γsecretingshowed that the specific T cell responses elicited by AE-TRIVN-CTB (1548±330 SFCs/106 splenocytes), AE-TRIVN-CTA(1642±514 SFCs/106 splenocytes), both regimen were significantly higher than AE-TRIVN(710.25±310 SFCs/106 splenocytes)(p<0.05).Taken together, these results showed that the prokaryotic expression recombinant plasmid of CTA and CTB gene were constructed successfully and detected its expression products in E.coli. Also, these results illustrated that the coincident expression of TRIVN and CTA or CTB has better immunity than the simply mixed regimen,which provided a good basis for further study on the adjuvantivity of cholera enterotoxin gene to DNA vaccine.