Cloning, Expression and Purification of a Novel Multi-epitopic HIV-1 Vaccine Candidate: A Preliminary Study on Immunoreactivity

Introduction: Designing an effective vaccine against human immunodeficiency virus (HIV)-1 is a global health priority. Multiepitope vaccines offer several potential advantages that may be promising in case of mutable divergent pathogens such as HIV-1. Herein, a multiepitopic recombinant protein containing various HIV-1 antigens was expressed in E. coli cells and its immunogenicity in combination with different adjuvants was initially evaluated in BALB/c mouse. Methods: HIVtop4 sequence spanning the junction of six amino acid fragments (Gag 158-186 , Pol 150-190 , ENV 296-323 , ENV 577-610 , Tat 1-20 and Tat 44-61 ) was designed based on immunoinformatic analysis to reduce the creation of junctional epitopes, improve the cleavage of proteasome and avoid the local accumulation of hydrophobic regions. Synthesized nucleotide sequence corresponding to HIVtop4 was cloned into pET23a plasmid. Expression of pET-HIVtop4 plasmid was induced in BL21 (DE3) E. coli cells by addition of 1 mM IPTG during 3 h culture and the protein was purified by Ni-NTA column chromatography and further confirmed against anti-His antibody in western-blotting. Groups of BALB/c mice (n=6) were immunized three times with 2 weeks interval, subcutaneously with 10 μg of candidate vaccine adjuvanted in Complete Freund’s adjuvant, Montanide ISA70 and Alum with suitable control groups. Two weeks after last immunization lymphocyte proliferation was measured with Brdu, IL-4 and IFN-γ cytokines with ELISA, total antibody and IgG1, IgG2a isotypes with indirect ELISA methods. Results: Results showed that Immunization with HIV-1 tat/ pol/gag/env led to a significant increase in the proliferative responses of lymphocytes, IL-4 and IFN- γ cytokine production and humoral immune response in comparison with the control groups. Conclusion: In this study we concluded that Tat, Env, Pol, Gag with adjuvants (Montanide, Alum and CFA) has potentials as a candidate vaccine against the HIV-1 virus. Vac Res, 2014, 1 (1): 10-15