Abstract
Background: HIV-1 Nef protein is a possible attractive target in the development of thera-peutic HIV vaccines including protein-based vaccines. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell-penetrating peptides (CPPs) as a carrier.Methods: In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-293T mammalian cell line using a novel cell-penetrating peptide, M918, in a non-covalent fashion. The size, zeta potential and morphology of the peptide/protein com-plexes were studied by scanning electron microscopy (SEM) and Zeta sizer. The efficiency of Nef and Hsp20-Nef transfection using M918 was evaluated by western blotting in HEK-293T cell line.Results: The SEM data confirmed the formation of discrete nanoparticles with a diameter of approxi-mately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively. The dominant band of ~ 27 kDa and ~ 47 kDa was detected in the transfected cells with the Nef/ M918 and Hsp20-Nef/ M918 nanoparticles at a molar ratio of 1:20 using anti-HIV-1 Nef monoclonal antibody. These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20-Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells.Conclusion: These data suggest that M918 CPP can be used to enter HIV-1 Nef and Hsp20-Nef pro-teins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.
Highlights
Human immunodeficiency virus (HIV) is an important public health issue
The scanning electron microscopy (SEM) data confirmed the formation of discrete nanoparticles with a diameter of approximately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively
These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells
Summary
Human immunodeficiency virus (HIV) is an important public health issue. In 2016, it was estimated that approximately 36.7 million people were living with a global HIV prevalence of 0.8% among adults [1]. It is of special importance to find novel and non-toxic CPPs with more extensive delivery properties. M918 peptide consists of 22 amino acids (MVTVLFRRLRIRRACGPPRVRV) derived from the Cterminal of the tumor suppressor protein p14ARF. This cationic CPP could be utilized for both covalent and noncovalent delivery of macromolecules in animal and plant cells [28]. We utilized HIV-1 Nef accessory protein and Nef linked to Hsp as two antigenic models for HIV-1 vaccine development in the future. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell-penetrating peptides (CPPs) as a carrier. Received: September 17, 2018 Revised: October 30, 2018 Accepted: December 02, 2018
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