Enhancing HIV-1 Nef Penetration into Mammalian Cells as an Antigen Candidate

Abstract

Introduction: The human immunodeficiency virus type 1 (HIV-1) Nef regulatory protein is known as a candidate for the design of therapeutic HIV DNA and protein vaccines. One of the limitations of these vaccines is the inability of DNA and protein to pass through the cell membrane. Various delivery systems have been developed to transfer DNA and protein into cells. Cell penetrating systems such as MPG and CyloP-1 are among delivery systems, which can deliver DNA and protein cargoes into the cells, respectively. Methods: In this study, we produced the recombinant Nef protein in Escherichia coli expression system. Then, the formation of CPP/DNA and CPP/protein nanoparticles was confirmed by agarose gel retardation, scanning electron microscope (SEM), Zetasizer and SDS-PAGE, and their stability was evaluated against nucleases and proteases. Finally, the delivery of the nanoparticles into HEK-293T cells was assessed by fluorescent microscopy, flow cytometry, and western blotting. Results: Our data confirmed the formation of stable nanoparticles through non-covalent bonds with a diameter of less than 200 nm. Moreover, the results of fluorescence microscopy, flow cytometry, and western blotting demonstrated that these CPPs could successfully deliver the Nef protein and DNA into HEK-293T cells. Conclusion: Our results indicated that the MPG and CyLoP-1 CPPs are suitable candidates for the delivery of DNA and protein cargoes into mammalian cells, respectively.

In vivo delivery of a multiepitope peptide and Nef protein using novel cell-penetrating peptides for development of HIV-1 vaccine candidate

“…MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV) (Morris 1997 ) and HR9 (CHHHHHRRRRRRRRRHHHHHC) (Liu et al 2015 ) peptides were mixed with pcDNA- nef-vpr-gp160-p24 or pcDNA- nef at N/P (nitrogen to phosphate) ratios of 10 and 5, respectively for nanoparticles formation as previously reported (Davoodi et al 2019a , b ). The size and charge of nanoparticles were assessed by scanning electron microscope (SEM) and Zetasizer at 25 °C, respectively.…”

“…In our previous study (Davoodi et al 2019a , b ), the Nef-Vpr-Gp160-P24 polyepitope peptide (/polypeptide) was designed by the conserved and immunogenic epitopes using bioinformatics analyses (Fig. 1 ).…”

“…The recombinant Nef-Vpr-Gp160-P24 polypeptide was expressed in the E. coli Rosetta strain transformed with pET-24a(+)- nef-vpr-gp160-p24 and purified by an affinity chromatography technique using Ni-NTA agarose column (Macherey-Nagel) under denaturing conditions as previously reported by our group (Davoodi et al 2019a , b ). To produce Nef protein, Escherichia coli Rosetta strain was transformed with pET-23a (+) harboring the nef gene.…”

“…To produce Nef protein, Escherichia coli Rosetta strain was transformed with pET-23a (+) harboring the nef gene. Nef protein was expressed and purified under native conditions as previously reported by our group (Davoodi et al 2019a , b ). In this study, the Nef-Vpr-Gp160-P24 polypeptide and Nef protein were produced in a large scale for mice immunization.…”

“…The aim of this study was the design of a novel polyepitope construct and evaluation of its immunogenicity in mice. In our previous study, the pEGFP- nef-vpr-gp160-p24 construct and the Nef-Vpr-Gp160-P24 polypeptide complexed with CPPs were designed for in vitro studies (Davoodi et al 2019a , b ). Herein, following in vitro studies, the pcDNA- nef-vpr-gp160-p24 and pcDNA- nef constructs, and the Nef-Vpr-Gp160-P24 polypeptide and Nef protein were prepared in large scale and endotoxin-free form.…”