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*Correspondence Email: A_bolhasani@pasteur.ac.ir Tel: +98 21 64112240 Fax: +98 21 66465132 Introduction: The Nef accessory protein is an attractive antigenic candidate in the development of HIV-1 DNAor protein-based vaccines. The most crucial disadvantage of DNA and protein-based vaccines is their low immunogenicity, which can be improved by cell-penetrating peptides (CPPs) as effective carrier molecules. Methods: In this study, the HIV-1 Nef protein was generated in the Escherichia coli expression system for in vitro delivery using a novel CPP, Latarcin 1 peptide, in a non-covalent manner. Also, the Histidine-rich nona-arginine peptide was utilized to transfer the HIV1 Nef gene. The size, morphology, and zeta potential of the complexes were evaluated by scanning electron microscopy (SEM) and Zetasizer. The efficiency of cell transfection was studied using a fluorescence microscopy and flow cytometry for the DNA/CPP complexes and western blot analysis for the protein/CPP complexes. Results: The Nef protein generated in the BL21 strain migrated as a dominant band of ~30 kDa in SDS-PAGE. The SEM data confirmed the formation of stable complexes with a size below 200 nm. MTT assay demonstrated that the complexes did not represent any considerable cytotoxic effect compared to untreated HEK-293T cells. The results of fluorescence microscopy, flow cytometry, and western blotting revealed that the Nef DNA and protein constructs could be significantly transfected into HEK-293T cell line using these CPPs. Conclusion: These data suggest that the Histidine-rich nonaarginine peptide and Latarcin 1 peptide as CPPs can be considered as a promising approach in the development of the HIV-1 vaccine for gene or protein delivery.

In vitro Delivery of HIV-1 Nef Antigen by Histidine-rich nona-arginine and Latarcin 1 peptide