BACKGROUND AND OBJECTIVES DNA-based therapeutic vaccines have been proposed as promising strategy for treatment of established HIV infections. However, these vaccines are often associated with certain shortcomings, such as poor… Click to show full abstract
BACKGROUND AND OBJECTIVES DNA-based therapeutic vaccines have been proposed as promising strategy for treatment of established HIV infections. However, these vaccines are often associated with certain shortcomings, such as poor immunogenicity and low transfection efficiency. In this study, we investigated ability of LL-37 to deliver a potential immunogenic fusion construct comprising HIV-1 nef and vpr genes into a mammalian cell line. METHODS First, the pEGFP-N1 eukaryotic expression vector harboring the HIV-1 nef-vpr fusion was produced free of endotoxin on large scale. Then, DNA/LL-37 complexes were prepared by co-incubation of pEGFP-nef-vpr with LL-37 for 45 minutes at different nitrogen to phosphate (N/P) ratios. Formation of DNA/peptide complexes was investigated by gel retardation assay. Next, stability and morphological characteristics of the nanoparticles were evaluated. Toxicity of LL-37 and the nanoparticles in HEK-293T cells was assessed by MTT assay. Transfection efficiency of the DNA/LL-37 complexes was studied by fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS LL-37 formed stable complexes with pEGFP-nef-vpr (diameter of 150-200 nm) while providing good protection against nucleolytic and proteolytic degradation. The peptide significantly affected cell viability even at low concentrations. However, the LL-37/DNA complexes had no significant cytotoxic effect. Treatment of cells with pEGFP-N1/LL-37 and pEGFP-nef-vpr/LL-37 resulted in transfection of 36.32% ± 1.13 and 25.55% ± 2.07 of cells, respectively. CONCLUSION Given these findings and the important immunomodulatory and antiviral activities of LL-37, the use of this peptide can be further exploited in the development of novel gene delivery strategies and vaccine design.