Title:In vitro Delivery of HIV-1 Nef-Vpr DNA Construct Using the Human
Antimicrobial Peptide LL-37
Volume: 19
Issue: 10
Author(s): Arash Nikyar, Azam Bolhassani*, Fatemeh Rouhollah*Masoumeh Heshmati
Affiliation:
- Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran
- Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences,
Islamic Azad University, Tehran, Iran
Keywords:
HIV-1, Nef, Vpr, LL-37, transfection, gene transfer techniques.
Abstract:
Background and Objectives: DNA-based therapeutic vaccines have been proposed as a
promising strategy for the 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 the 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 a large scale. Then, DNA/LL-37 complexes were prepared by coincubation
of pEGFP-nef-vpr with LL-37 for 45 minutes at different nitrogen to phosphate (N/P) ratios.
The formation of DNA/peptide complexes was investigated by gel retardation assay. Next, the stability
and morphological characteristics of the nanoparticles were evaluated. The toxicity of LL-37 and the
nanoparticles in HEK-293T cells were assessed by MTT assay. The 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.
