en Other Other Original article Original article <strong>Introduction: </strong>The Nef accessory protein is an attractive antigenic candidate in the development of HIV-1 DNA- or 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. <strong>Methods: </strong>In this study, the HIV-1 Nef protein was generated in the <em>Escherichia coli </em>expression system for <em>in vitro</em> 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 HIV-1 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. <strong>Results: </strong>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. <strong>Conclusion: </strong>These data suggest that the Histidine-rich nona-arginine 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.&nbsp; Therapeutic vaccine, HIV-1 Nef, Cell-penetrating peptides, Transfection 107 115 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-123-4&slc_lang=en&sid=1 Fatemeh Namazi 0000-0003-4611-855X No Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran Azam Bolhassani azam.bolhassani@yahoo.com 0000000173637406 Yes Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran Seyed Mehdi Sadat 0000-0001-7739-179X No Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran Shiva Irani 0000-0002-4202-9931 No Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran