Delivery of HIV-1 Polyepitope Constructs Using Cationic and Amphipathic Cell Penetrating Peptides into Mammalian Cells.

Background: An effective vaccine against human immunodeficiency virus 1 (HIV-1) is an important global health priority. Despite many efforts in the development of the HIV-1 vaccine, no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic and conserved epitopes of HIV-1 proteins have received special attention.

Methods: In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for selection of immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis. At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was designed using in silico studies. Then, the DNA construct was subcloned in pEGFP-N1 and pET- 24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coli expression system for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA construct.

Results: Our results showed that the recombinant polyepitope peptide generated in Rosetta strain migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent any considerable cytotoxic effect compared to untreated cells. The results of fluorescence microscopy, flow cytometry and western blotting indicated that these CPPs successfully delivered polyepitope constructs into HEK-293T cell line.

Conclusion: These data suggested that these CPPs can be used as a promising approach for the development of the HIV-1 vaccine.