From simulation to immunization: efficient non-viral delivery of HIV-1 Nef^mut-Tat DNA construct using CM18-TAT11 cell...

DNA vaccination has been suggested as an attractive therapeutic modality for many diseases. However, their immunogenicity remains low due to poor transfection and antigen production. To address this limitation, we investigated the CM18-TAT11 cell-penetrating peptide (CPP) as a delivery system for an HIV-1 Nef^mut-Tat DNA vaccine construct (pDNA). At first, the CM18-TAT11 peptide properties, structural models, and interaction with DNA were evaluated using different web servers and molecular dynamics (MD) simulations. Then, the pDNA/CM18-TAT11 complexes were prepared at different nitrogen-to-phosphate (N/P) ratios and the best ratio was determined according to migration on the gel, stability and physicochemical properties (nanosize and surface charge) for delivery into mammalian cells. Next, transfection efficiency of the nanoparticles was assessed using GFP reporter via flow cytometry and fluorescence microscopy. Finally, after mice immunization with the pcDNA-Nef^mut-Tat/CM18-TAT11 complexes at the optimal N/P ratio, antigen-specific immune responses were evaluated through antibody responses, cytokine profiles, and cytotoxic T lymphocyte (CTL) activity. Bioinformatics analysis confirmed favorable properties of CM18-TAT11 including high cell-penetrating probability, minimal toxicity, and low immunogenicity. MD simulations revealed that interaction of CM18-TAT11 with DNA saturated rather quickly, characterized by two distinct binding patterns. In vitro analysis showed that CM18-TAT11 effectively condensed pDNA into nano-sized particles that induced no cytotoxicity and facilitated delivery of the plasmid into HEK-293 T cells at an N/P ratio of 2:1. In vivo analysis indicated that the pDNA/CM18-TAT11 nanoparticles significantly elicited robust Th1 and CTL-mediated cellular immunity. Taken together, these results demonstrated the potential of CM18-TAT11 as a promising gene delivery system for HIV-1 therapeutic DNA vaccine development.