Investigation of Immunostimulatory Effects of IFN‐γ Cytokine and CD40 Ligand Costimulatory Molecule for Development of HIV‐1 Therapeutic Vaccine Candidate

The most crucial disadvantage of DNA‐based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV‐1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single‐cycle replicable (SCR) HIV‐1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th‐1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ‐Nef and CD40L‐Nef fusion DNA constructs show higher secretion of IFNγ and TNF‐α from virion‐infected lymphocytes than other groups. Therefore, the IFNγ‐Nef and CD40L‐Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV‐1 vaccine. This manuscript shows that interferon Gamma (IFNγ) or CD40 ligand (CD40L) adjuvants can effectively induce Nef‐specific long‐term immune responses especially when adjuvants are linked to the N‐terminal region of the Nef antigen. Moreover, the fusion constructs can considerably stimulate the secretion of IFN‐γ and tumor necrosis factor alpha (TNF‐α) in the infected lymphocytes with single‐cycle replicable human immunodeficiency virus (SCR HIV‐1) virions in vitro.