Delivery of HIV-1 Nef linked to heat shock protein 27 using a cationic polymer is more effective than cationic lipid in mammalian cells

Milani, Alireza; Bolhassani, Azam; Heshmati, Masoumeh

doi:10.4149/bll_2017_064

Cited by 8 publications

(13 citation statements)

References 25 publications

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“…The full length Nef gene (from HIV‐1 vector pNL4‐3, Accession No: AF324493.2, ∼648) was previously cloned into pcDNA3.1 (−) and pEGFP‐N1 expression vectors . To construct the pcDNA‐Hsp20‐Nef and pEGFP‐Hsp20‐Nef, the fusion Hsp20‐Nef construct was subcloned from pET‐Hsp20‐Nef (previously provided by our group) into the pcDNA3.1 and pEGFP‐N1 using Bam HI/ Hind III and Bgl II/ Hind III restriction enzymes, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The full length Nef gene (from HIV-1 vector pNL4-3, Accession No: AF324493.2, 648) was previously cloned into pcDNA3.1 (−) and pEGFP-N1 expression vectors. 18,22 To construct the pcDNA-Hsp20-Nef and pEGFP-Hsp20-Nef, the fusion Hsp20-Nef construct was subcloned from pET-Hsp20-Nef (previously provided by our group 21 ) into the pcDNA3.1 and pEGFP-N1 using BamHI/ HindIII and BglII/HindIII restriction enzymes, respectively. Finally, all DNA constructs containing Nef and Hsp20-Nef fusion (i.e., pEGFP-Nef, pcDNA-Nef, pEGFP-Hsp20-Nef & pcDNA-Hsp20-Nef) were purified by an Endo-free plasmid Mega kit (Qiagen, Germany).…”

Section: Generation Of the Recombinant Dna Constructsmentioning

confidence: 99%

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Gene and protein delivery using four cell penetrating peptides for HIV‐1 vaccine development

et al. 2019

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Cell penetrating peptides (CPPs) can potently transport therapeutic molecules to target cells for treatment of a variety of diseases. Thus, their use is critical to improve therapeutic vaccines. Histidine‐rich nona‐arginine (HR9) and primary amphipathic peptide (MPG) showed the ability to transfer DNA into the cells. Moreover, the peptide derived from the C‐terminal of the tumor suppressor protein p14ARF (M918) and arginine‐rich peptide (penetratin) were utilized to deliver polypeptides and proteins into the living cells. In this study, the immunostimulatory properties of HIV‐1 Nef DNA and protein constructs were evaluated using small heat shock protein 20 (sHsp20) and Freund's emulsion as an adjuvant, and four CPPs (HR9, MPG, M918, and penetratin) as a gene or protein carrier in BALB/c mice. Our data indicated that the HR9/DNA, MPG/DNA, M918/protein, and penetratin/protein complexes formed the stable nanoparticles that were effectively delivered in HEK‐293T cell line at certain ratios. Moreover, a heterologous Hsp20‐Nef DNA + MPG prime/rHsp20‐Nef protein+M918 boost regimen significantly elicited higher levels of IgG2a, IgG2b, IFN‐gamma, and Granzyme B directed toward Th1 responses in a long period (3 months) after the last immunization compared to other groups. Furthermore, the effective role of Hsp20 was detected as a natural adjuvant in enhancing immune responses against HIV‐1 Nef antigen. These findings demonstrated that the simultaneous use of M918 and MPG CPPs as protein and gene carriers improves HIV‐1 Nef‐specific B‐ and T‐cell immune responses as a promising approach for development of HIV‐1 monovalent vaccine.

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Section: Methodsmentioning

confidence: 99%

Section: Generation Of the Recombinant Dna Constructsmentioning

confidence: 99%

Gene and protein delivery using four cell penetrating peptides for HIV‐1 vaccine development

et al. 2019

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“…The nef, hsp27 and hsp27-nef genes were previously confirmed as clear bands of ~648 bp, ~720 bp and ~1368 bp on agarose gel [13]. The pEGFP-nef, pEGFP-hsp27 and pEGFP-hsp27-nef, pEGFP-hsp70 and pEGFP-hsp70-nef were prepared with high purity.…”

Section: Generation Of the Recombinant Dna Constructsmentioning

confidence: 83%

“…To monitor the efficiency of transfection, pEGFP-N1 and pEGFP-N3 eukaryotic vectors encoding enhanced green fluorescent protein (EGFP) were employed in this study. The constructions of the full length pEGFP-nef, pEGFP-hsp27 and pEGFP-hsp27-nef genes were described in our previous study [13]. Moreover, the full length of hsp70 gene was prepared in a pUC57 cloning vector by Biomatik Company (Canada).…”

Section: Preparation Of the Recombinant Plasmidsmentioning

confidence: 99%

The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells

Milani

Rouhollah

Naseroleslami

et al. 2020

vacres

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Introduction: Vaccine design is mainly considered as a therapeutic strategy to elicit HIVspecific immunity. DNA vaccines encoding an antigen and also an adjuvant can induce an effective adaptive immunity. Due to having numerous roles in viral infection, the HIV-1 Nef protein is considered as an antigen candidate for development of therapeutic vaccines. A variety of adjuvants and delivery systems have been utilized to increase the potency of DNA vaccines against viral infections, such as heat shock proteins (HSPs) which possess chaperon activity and immunostimulatory properties. Methods: pEGFP mammalian expression vectors harboring nef, hsp27, hsp27-nef, hsp70 and hsp70-nef genes were prepared in large scale. Their concentration and purity were assessed by NanoDrop spectrophotometry. Human embryonic kidney 293T cells (HEK-293T) were grown in DMEM culture medium and transfected with these constructs using Lipofectamine 2000 transfection reagent. After 48 hours, their transfection efficiency was evaluated using fluorescent microscopy and flow cytometry. Results: The pEGFP-nef,-hsp27,-hsp27-nef,-hsp70 and-hsp70-nef constructs were successfully prepared in large scale and high purity. The results of cell transfection with each construct showed that the percentages of Nef

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“…The Nef protein has major roles including inhibition of CD4 activity, stimulation of virus replication, and induction of apoptosis in both uninfected and infected cells [4]. Generally, the advantages of HIV-1 Nef as a good antigen candidate for HIV vaccine development include its expression in the first step of the infection, its presence during the period of viral infection, and possessing T-cell epitopes [5-10]. An effective vaccine should induce antibody response along with an increased cellular immunity against the viral infection.…”

Section: Introductionmentioning

confidence: 99%

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

Rostami

Irani

Bolhassani

et al. 2019

CHR

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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.

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Delivery of HIV-1 Nef linked to heat shock protein 27 using a cationic polymer is more effective than cationic lipid in mammalian cells

Abstract: Generally, Hsp27 can be used as a suitable carrier in DNA vaccine design against HIV-1 infections (Fig. 5, Ref. 28).

Cited by 8 publications

References 25 publications

Gene and protein delivery using four cell penetrating peptides for HIV‐1 vaccine development

Gene and protein delivery using four cell penetrating peptides for HIV‐1 vaccine development

The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells

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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