Comparison of HIV-1 Vif and Vpu accessory proteins for delivery of polyepitope constructs harboring Nef, Gp160 and P24 using various cell penetrating peptides

Abstract

To develop an effective therapeutic vaccine against HIV-1, prediction of the most conserved epitopes derived from major proteins using bioinformatics tools is an alternative achievement. The epitope-driven vaccines against variable pathogens represented successful results. Hence, to overcome this hyper-variable virus, we designed the highly conserved and immunodominant peptide epitopes. Two servers were used to predict peptide-MHC-I binding affinity including NetMHCpan4.0 and Syfpeithi servers. The NetMHCIIpan3.2 server was utilized for MHC-II binding affinity. Then, we determined immunogenicity scores and allergenicity by the IEDB immunogenicity predictor and Algpred, respectively. Next, for estimation of toxicity and population coverage, ToxinPred server and IEDB population coverage tool were applied. After that, the MHC-peptide binding was investigated by GalexyPepDock peptide-protein flexible docking server. Finally, two different DNA and peptide constructs containing Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 were prepared and complexed with four various cell penetrating peptides (CPPs) for delivery into mammalian cells (MPG and HR9 CPPs for DNA delivery, and CyLoP-1 and LDP-NLS CPPs for protein delivery). Our results indicated that the designed DNA and peptide constructs could form non-covalent stable nanoparticles at certain ratios as observed by scanning electron microscope (SEM) and Zetasizer. The flow cytometry results obtained from in vitro transfection of the nanoparticles into HEK-293T cell lines showed that the percentage of GFP expressing cells was about 38.38 ± 1.34%, 25.36% ± 0.30, 54.95% ± 0.84, and 25.11% ± 0.36 for MPG/pEGFP-nef-vif-gp160-p24, MPG/pEGFP-nef-vpu-gp160-p24, HR9/pEGFP-nef-vif-gp160-p24 and HR9/pEGFP-nef-vpu-gp160-p24, respectively. Thus, these data showed that the DNA construct harboring nef-vif-gp160-p24 multi-epitope gene had higher efficiency than the DNA construct harboring nef-vpu-gp160-p24 multi-epitope gene to penetrate into the cells. Moreover, delivery of the recombinant Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 polyepitope peptides in HEK-293T cells was confirmed as a single band about 32 kDa using western blot analysis. Although, both DNA and peptide constructs could be successfully transported by a variety of CPPs into the cells, but the difference between them in transfection rate will influence the levels of immune responses for development of therapeutic vaccines.

Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection

“…Long peptides have demonstrated superior efficacy in eliciting immune responses compared to short peptides [35]. However, there remains uncertainty regarding whether artificially anticipated multiple-epitope constructs, comprising T-and B-cell epitopes, can be adequately displayed and processed to activate targeted immunity.…”

Portadores do vírus da imunodeficiência humana não progressores a longo prazo

“…O vírus da imunodeficiência humana (HIV) é um retrovírus de RNA, formado por três genes estruturais gag, env e pol comuns aos retrovírus, e ainda, genes regulatórios tat, rev e nef, e os genes acessórios vif, vpr e vpu ou vpx, que tem como as principais células alvo, aquelas que expressam CD4 em sua superfície, ganhando destaque os linfócitos T (KARDANI et al, 2019).…”

Neoantigen polypeptide vaccines induce effective antitumor response in colorectal cancer

“…First, 8-11amino-acid long mutant peptides were generated for MHC I-restricted neoantigen prediction, and 12-15-amino-acid long mutant peptides were generated for MHC II-restricted neoantigen prediction. Then HLA binding affinity for each peptide was calculated by NetMHCpan4.0(16) and NetMHCIIpan3.2 (17) for MHC I and MHC II molecules respectively. Expression level of mutant genes was derived from RNAseq data in transcripts per million (TPM).…”