Dataset Information

M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

ABSTRACT: BACKGROUND:HIV-1 Nef protein is a possible attractive target in the development of therapeutic 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 complexes 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 approximately 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 proteins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.

SUBMITTER: Rostami B

PROVIDER: S-EPMC6416460 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

Rostami Bahareh B Irani Shiva S Bolhassani Azam A Cohan Reza Ahangari RA

Current HIV research 20180101 4

<h4>Background</h4>HIV-1 Nef protein is a possible attractive target in the development of therapeutic 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.<h4>Methods</h4>In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-29 ...[more]

PMID: 30520377

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Project description: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.

Dataset Information

M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

Publications

M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

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