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Current HIV Research

Author(s): Fatemeh Namazi, Azam Bolhassani*, Seyed Mehdi Sadat and Shiva Irani

DOI: 10.2174/1570162X17666191121114522

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Delivery of HIV-1 Polyepitope Constructs Using Cationic and Amphipathic Cell Penetrating Peptides into Mammalian Cells

Page: [408 - 428] Pages: 21

  • * (Excluding Mailing and Handling)

Abstract

Background: An effective vaccine against human immunodeficiency virus 1 (HIV-1) is an important global health priority. Despite many efforts in the development of the HIV-1 vaccine, no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic and conserved epitopes of HIV-1 proteins have received special attention.

Methods: In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for selection of immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis. At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was designed using in silico studies. Then, the DNA construct was subcloned in pEGFP-N1 and pET- 24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coli expression system for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA construct.

Results: Our results showed that the recombinant polyepitope peptide generated in Rosetta strain migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent any considerable cytotoxic effect compared to untreated cells. The results of fluorescence microscopy, flow cytometry and western blotting indicated that these CPPs successfully delivered polyepitope constructs into HEK-293T cell line.

Conclusion: These data suggested that these CPPs can be used as a promising approach for the development of the HIV-1 vaccine.

Keywords: HIV-1, cell-penetrating peptides, transfection, bioinformatics studies, DNA construct, Peptide construct.

Graphical Abstract

[1]
Alves BM, Siqueira JD, Prellwitz IM, et al. Estimating HIV-1 genetic diversity in Brazil through next-generation sequencing. Front Microbiol 2019; 10: 749.
[http://dx.doi.org/10.3389/fmicb.2019.00749] [PMID: 31024510]
[2]
Seitz R. Human immunodeficiency virus (HIV). Transfus Med Hemother 2016; 43(3): 203-22.
[http://dx.doi.org/10.1159/000445852] [PMID: 27403093]
[3]
Rambaut A, Posada D, Crandall KA, Holmes EC. The causes and consequences of HIV evolution. Nat Rev Genet 2004; 5(1): 52-61.
[http://dx.doi.org/10.1038/nrg1246] [PMID: 14708016]
[4]
Li G, Piampongsant S, Faria NR, et al. An integrated map of HIV genome-wide variation from a population perspective. Retrovirology 2015; 12(1): 18.
[http://dx.doi.org/10.1186/s12977-015-0148-6] [PMID: 25808207]
[5]
Khairkhah N, Namvar A, Kardani K, Bolhassani A. Prediction of cross-clade HIV-1 T-cell epitopes using immunoinformatics analysis. Proteins 2018; 86(12): 1284-93.
[http://dx.doi.org/10.1002/prot.25609] [PMID: 30260061]
[6]
Joseph AM, Ladha JS, Mojamdar M, Mitra D. Human immunodeficiency virus-1 Nef protein interacts with Tat and enhances HIV-1 gene expression. FEBS Lett 2003; 548(1-3): 37-42.
[http://dx.doi.org/10.1016/S0014-5793(03)00725-7] [PMID: 12885404]
[7]
Quaranta MG, Mattioli B, Giordani L, Viora M. Immunoregulatory effects of HIV-1 Nef protein. Biofactors 2009; 35(2): 169-74.
[http://dx.doi.org/10.1002/biof.28] [PMID: 19449444]
[8]
Pandey RK, Ojha R, Aathmanathan VS, Krishnan M, Prajapati VK. Immunoinformatics approaches to design a novel multi-epitope subunit vaccine against HIV infection. Vaccine 2018; 36(17): 2262-72.
[http://dx.doi.org/10.1016/j.vaccine.2018.03.042] [PMID: 29571972]
[9]
Felli C, Vincentini O, Silano M, Masotti A. HIV-1 Nef signaling in intestinal mucosa epithelium suggests the existence of an active inter-kingdom crosstalk mediated by exosomes. Front Microbiol 2017; 8: 1022.
[http://dx.doi.org/10.3389/fmicb.2017.01022] [PMID: 28642743]
[10]
Jazaeri EO, Mahdavi A, Abdoli A. Formulation of chitosan with the polyepitope HIV-1 protein candidate vaccine efficiently boosts cellular immune responses in mice. Pathog Dis 2017; 75(8)
[http://dx.doi.org/10.1093/femspd/ftx098] [PMID: 28911033]
[11]
Bolesta E, Gzyl J, Wierzbicki A, et al. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens. Virology 2005; 332(2): 467-79.
[http://dx.doi.org/10.1016/j.virol.2004.09.043] [PMID: 15680412]
[12]
Zuñiga R, Lucchetti A, Galvan P, et al. Relative dominance of Gag p24-specific cytotoxic T lymphocytes is associated with human immunodeficiency virus control. J Virol 2006; 80(6): 3122-5.
[http://dx.doi.org/10.1128/JVI.80.6.3122-3125.2006] [PMID: 16501126]
[13]
Koff WC. HIV vaccine development: challenges and opportunities towards solving the HIV vaccine-neutralizing antibody problem. Vaccine 2012; 30(29): 4310-5.
[http://dx.doi.org/10.1016/j.vaccine.2011.11.014] [PMID: 22100891]
[14]
Koff WC, Russell ND, Walport M, et al. Accelerating the development of a safe and effective HIV vaccine: HIV vaccine case study for the Decade of Vaccines. Vaccine 2013; 31(Suppl. 2): B204-8.
[http://dx.doi.org/10.1016/j.vaccine.2012.10.115] [PMID: 23598483]
[15]
Karpenko LI, Bazhan SI, Antonets DV, Belyakov IM. Novel approaches in polyepitope T-cell vaccine development against HIV-1. Expert Rev Vaccines 2014; 13(1): 155-73.
[http://dx.doi.org/10.1586/14760584.2014.861748] [PMID: 24308576]
[16]
Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK. Peptide vaccine: Progress and challenges. Vaccines (Basel) 2014; 2(3): 515-36.
[http://dx.doi.org/10.3390/vaccines2030515] [PMID: 26344743]
[17]
Rosa DS, Ribeiro SP, Fonseca SG, et al. Multiple approaches for increasing the immunogenicity of an epitope-based anti-HIV vaccine. AIDS Res Hum Retroviruses 2015; 31(11): 1077-88.
[http://dx.doi.org/10.1089/aid.2015.0101] [PMID: 26149745]
[18]
Alhakamy NA, Nigatu AS, Berkland CJ, Ramsey JD. Noncovalently associated cell-penetrating peptides for gene delivery applications. Ther Deliv 2013; 4(6): 741-57.
[http://dx.doi.org/10.4155/tde.13.44] [PMID: 23738670]
[19]
Farkhani SM, Valizadeh A, Karami H, Mohammadi S, Sohrabi N, Badrzadeh F. Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules. Peptides 2014; 57: 78-94.
[http://dx.doi.org/10.1016/j.peptides.2014.04.015] [PMID: 24795041]
[20]
Madani F, Lindberg S, Langel U, Futaki S, Gräslund A. Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011; 2011 414729
[http://dx.doi.org/10.1155/2011/414729] [PMID: 21687343]
[21]
Bolhassani A. Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochim Biophys Acta 2011; 1816(2): 232-46.
[http://dx.doi.org/10.1016/j.bbcan.2011.07.006] [PMID: 21840374]
[22]
Bolhassani A, Jafarzade BS, Mardani G. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides. Peptides 2017; 87: 50-63.
[http://dx.doi.org/10.1016/j.peptides.2016.11.011] [PMID: 27887988]
[23]
Hoffmann K, Milech N, Juraja SM, et al. A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery. Sci Rep 2018; 8(1): 12538.
[http://dx.doi.org/10.1038/s41598-018-30790-2] [PMID: 30135446]
[24]
Rostami B, Irani S, Bolhassani A, Cohan RA. M918: A novel cell penetrating peptide for effective delivery of HIV-1 Nef and Hsp20-Nef proteins into eukaryotic cell lines. Curr HIV Res 2018; 16(4): 280-7.
[http://dx.doi.org/10.2174/1570162X17666181206111859] [PMID: 30520377]
[25]
Shahbazi S, Bolhassani A. Comparison of six cell penetrating peptides with different properties for in vitro and in vivo delivery of HPV16 E7 antigen in therapeutic vaccines. Int Immunopharmacol 2018; 62: 170-80.
[http://dx.doi.org/10.1016/j.intimp.2018.07.006] [PMID: 30015237]
[26]
Mussbach F, Franke M, Zoch A, Schaefer B, Reissmann S. Transduction of peptides and proteins into live cells by cell penetrating peptides. J Cell Biochem 2011; 112(12): 3824-33.
[http://dx.doi.org/10.1002/jcb.23313] [PMID: 21826709]
[27]
Kristensen M, Birch D, Mørck Nielsen H. Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos. Int J Mol Sci 2016; 17(2): 185.
[http://dx.doi.org/10.3390/ijms17020185] [PMID: 26840305]
[28]
Tashima T. Intelligent substance delivery into cells using cell-penetrating peptides. Bioorg Med Chem Lett 2017; 27(2): 121-30.
[http://dx.doi.org/10.1016/j.bmcl.2016.11.083] [PMID: 27956345]
[29]
Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ. Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials 2011; 32(13): 3520-37.
[http://dx.doi.org/10.1016/j.biomaterials.2011.01.041] [PMID: 21329975]
[30]
Morris MC, Deshayes S, Heitz F, Divita G. Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell 2008; 100(4): 201-17.
[http://dx.doi.org/10.1042/BC20070116] [PMID: 18341479]
[31]
Ponnappan N, Chugh A. Cell-penetrating and cargo-delivery ability of a spider toxin-derived peptide in mammalian cells. Eur J Pharm Biopharm 2017; 114: 145-53.
[http://dx.doi.org/10.1016/j.ejpb.2017.01.012] [PMID: 28159722]
[32]
Ponnappan N, Budagavi DP, Chugh A. CyLoP-1: Membrane-active peptide with cell-penetrating and antimicrobial properties. Biochim Biophys Acta Biomembr 2017; 1859(2): 167-76.
[http://dx.doi.org/10.1016/j.bbamem.2016.11.002] [PMID: 27836642]
[33]
Moret I, Esteban Peris J, Guillem VM, et al. Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum. J Control Release 2001; 76(1-2): 169-81.
[http://dx.doi.org/10.1016/S0168-3659(01)00415-1] [PMID: 11532322]
[34]
Saleh T, Bolhassani A, Shojaosadati SA, Hosseinkhani S. Evaluation of cell penetrating peptide delivery system on HPV16E7 expression in three types of cell line. Iranian J Biotechnol 2015; 13(1): 55-62.
[http://dx.doi.org/10.15171/ijb.1115] [PMID: 28959282]
[35]
Mahdavi M, Ebtekar M, Azadmanesh K, et al. HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model. Acta Virol 2010; 54(2): 131-6.
[http://dx.doi.org/10.4149/av_2010_02_131] [PMID: 20545443]
[36]
Mahdavi M, Ebtekar M, Mahboudi F, et al. Immunogenicity of a new HIV-1 DNA construct in a BALB/c mouse model. Iran J Immunol 2009; 6(4): 163-73.
[PMID: 20054104]
[37]
Mothe B, Llano A, Ibarrondo J, et al. Definition of the viral targets of protective HIV-1-specific T cell responses. J Transl Med 2011; 9(1): 208.
[http://dx.doi.org/10.1186/1479-5876-9-208] [PMID: 22152067]
[38]
Vardas E, Stanescu I, Leinonen M, et al. Indicators of therapeutic effect in FIT-06, a Phase II trial of a DNA vaccine, GTU(®)-Multi-HIVB, in untreated HIV-1 infected subjects. Vaccine 2012; 30(27): 4046-54.
[http://dx.doi.org/10.1016/j.vaccine.2012.04.007] [PMID: 22549090]
[39]
Reguzova A, Antonets D, Karpenko L, Ilyichev A, Maksyutov R, Bazhan S. Design and evaluation of optimized artificial HIV-1 poly-T cell-epitope immunogens. PLoS One 2015; 10(3) e0116412
[http://dx.doi.org/10.1371/journal.pone.0116412] [PMID: 25786238]
[40]
Ahmed T, Borthwick NJ, Gilmour J, Hayes P, Dorrell L, Hanke T. Control of HIV-1 replication in vitro by vaccine-induced human CD8(+) T cells through conserved subdominant Pol epitopes. Vaccine 2016; 34(9): 1215-24.
[http://dx.doi.org/10.1016/j.vaccine.2015.12.021] [PMID: 26784683]
[41]
Kurcinski M, Jamroz M, Blaszczyk M, Kolinski A, Kmiecik S. CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site. Nucleic Acids Res 2015; 43(W1): W419-24.
[http://dx.doi.org/10.1093/nar/gkv456] [PMID: 25943545]
[42]
Blaszczyk M, Kurcinski M, Kouza M, et al. Modeling of protein-peptide interactions using the CABS-dock web server for binding site search and flexible docking. Methods 2016; 93: 72-83.
[http://dx.doi.org/10.1016/j.ymeth.2015.07.004] [PMID: 26165956]
[43]
Ciemny MP, Kurcinski M, Blaszczyk M, Kolinski A, Kmiecik S. Modeling EphB4-EphrinB2 protein-protein interaction using flexible docking of a short linear motif. Biomed Eng Online 2017; 16(Suppl. 1): 71.
[http://dx.doi.org/10.1186/s12938-017-0362-7] [PMID: 28830442]
[44]
Skwarczynski M, Toth I. Peptide-based synthetic vaccines. Chem Sci (Camb) 2016; 7(2): 842-54.
[http://dx.doi.org/10.1039/C5SC03892H] [PMID: 28791117]
[45]
Rathnayake PV, Gunathunge BG, Wimalasiri PN, Karunaratne DN, Ranatunga RJ. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study. J Biophys 2017; 2017 1059216
[http://dx.doi.org/10.1155/2017/1059216] [PMID: 28321253]
[46]
Keller AA, Mussbach F, Breitling R, et al. Relationships between cargo, cell penetrating peptides and cell type for uptake of non-covalent complexes into live cells. Pharmaceuticals (Basel) 2013; 6(2): 184-203.
[http://dx.doi.org/10.3390/ph6020184] [PMID: 24275947]
[47]
Durzyńska J, Przysiecka Ł, Nawrot R, et al. Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine. J Pharmacol Exp Ther 2015; 354(1): 32-42.
[http://dx.doi.org/10.1124/jpet.115.223305] [PMID: 25922342]
[48]
Liu BR, Lin MD, Chiang HJ, Lee HJ. Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene 2012; 505(1): 37-45.
[http://dx.doi.org/10.1016/j.gene.2012.05.053] [PMID: 22669044]
[49]
Dai YH, Liu BR, Chiang HJ, Lee HJ. Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium. Gene 2011; 489(2): 89-97.
[http://dx.doi.org/10.1016/j.gene.2011.08.011] [PMID: 21925248]
[50]
Chen YJ, Liu BR, Dai YH, et al. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 2012; 493(2): 201-10.
[http://dx.doi.org/10.1016/j.gene.2011.11.060] [PMID: 22173105]
[51]
Liu BR, Liou JS, Chen YJ, Huang YW, Lee HJ. Delivery of nucleic acids, proteins, and nanoparticles by arginine-rich cell-penetrating peptides in rotifers. Mar Biotechnol (NY) 2013; 15(5): 584-95.
[http://dx.doi.org/10.1007/s10126-013-9509-0] [PMID: 23715807]
[52]
Mandraccia L, Slavin G. Cell membrane. Hauppauge: Nova Science Publishers, Inc. 2013.
[53]
Crombez L, Morris MC, Deshayes S, Heitz F, Divita G. Peptide-based nanoparticle for ex vivo and in vivo drug delivery. Curr Pharm Des 2008; 14(34): 3656-65.
[http://dx.doi.org/10.2174/138161208786898842] [PMID: 19075741]
[54]
Eaton P, Quaresma P, Soares C, et al. A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles. Ultramicroscopy 2017; 182: 179-90.
[http://dx.doi.org/10.1016/j.ultramic.2017.07.001] [PMID: 28692935]
[55]
Morris MC, Gros E, Aldrian-Herrada G, et al. A non-covalent peptide-based carrier for in vivo delivery of DNA mimics. Nucleic Acids Res 2007; 35(7) e49
[http://dx.doi.org/10.1093/nar/gkm053] [PMID: 17341467]
[56]
Rádis-Baptista G, Campelo IS, Morlighem JRL, Melo LM, Freitas VJF. Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis. J Biotechnol 2017; 252: 15-26.
[http://dx.doi.org/10.1016/j.jbiotec.2017.05.002] [PMID: 28479163]
[57]
Künnapuu K, Veiman KL, Porosk L, et al. Tumor gene therapy by systemic delivery of plasmid DNA with cell‐penetrating peptides. FASEB BioAdvances 2019; 1: 105-14.
[http://dx.doi.org/10.1096/fba.1026]
[58]
Liu BR, Huang YW, Aronstam RS, Lee HJ. Identification of a short cell-penetrating peptide from bovine lactoferricin for intracellular delivery of DNA in human A549 cells. PLoS One 2016; 11(3) e0150439
[http://dx.doi.org/10.1371/journal.pone.0150439] [PMID: 26942714]
[59]
Huang YW, Lee HJ, Tolliver LM, Aronstam RS. Delivery of nucleic acids and nanomaterials by cell-penetrating peptides: opportunities and challenges. BioMed Res Int 2015; 2015834079
[http://dx.doi.org/10.1155/2015/834079] [PMID: 25883975]
[60]
Dubovskii PV, Vassilevski AA, Kozlov SA, Feofanov AV, Grishin EV, Efremov RG. Latarcins: versatile spider venom peptides. Cell Mol Life Sci 2015; 72(23): 4501-22.
[http://dx.doi.org/10.1007/s00018-015-2016-x] [PMID: 26286896]
[61]
Kuhn-Nentwig L. Antimicrobial and cytolytic peptides of venomous arthropods. Cell Mol Life Sci 2003; 60(12): 2651-68.
[http://dx.doi.org/10.1007/s00018-003-3106-8] [PMID: 14685689]
[62]
Jha D, Mishra R, Gottschalk S, et al. CyLoP-1: a novel cysteine-rich cell-penetrating peptide for cytosolic delivery of cargoes. Bioconjug Chem 2011; 22(3): 319-28.
[http://dx.doi.org/10.1021/bc100045s] [PMID: 21319732]
[63]
Pereira HA, Tsyshevskaya-Hoover I, Hinsley H, et al. Candidacidal activity of synthetic peptides based on the antimicrobial domain of the neutrophil-derived protein, CAP37. Med Mycol 2010; 48(2): 263-72.
[http://dx.doi.org/10.3109/13693780903081976] [PMID: 19626550]
[64]
Laufer SD, Restle T. Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems. Curr Pharm Des 2008; 14(34): 3637-55.
[http://dx.doi.org/10.2174/138161208786898806] [PMID: 19075740]
[65]
Patel SG, Sayers EJ, He L, et al. Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Sci Rep 2019; 9(1): 6298.
[http://dx.doi.org/10.1038/s41598-019-42456-8] [PMID: 31000738]
[66]
Silva S, Almeida AJ, Vale N. Combination of cell-penetrating peptides with nanoparticles for therapeutic application: A Review. Biomolecules 2019; 9(1) E22
[http://dx.doi.org/10.3390/biom9010022] [PMID: 30634689]
[67]
Allolio C, Magarkar A, Jurkiewicz P, et al. Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore. Proc Natl Acad Sci USA 2018; 115(47): 11923-8.
[http://dx.doi.org/10.1073/pnas.1811520115] [PMID: 30397112]
[68]
Jiang Y, Li M, Zhang Z, Gong T, Sun X. Cell-penetrating peptides as delivery enhancers for vaccine. Curr Pharm Biotechnol 2014; 15(3): 256-66.
[http://dx.doi.org/10.2174/1389201015666140813130114] [PMID: 25142954]
[69]
Yang J, Luo Y, Shibu MA, Toth I, Skwarczynskia M. Cell-penetrating peptides: Efficient vectors for vaccine delivery. Curr Drug Deliv 2019; 16(5): 430-43.
[http://dx.doi.org/10.2174/1567201816666190123120915] [PMID: 30760185]
[70]
Skwarczynski M, Toth I. Cell-penetrating peptides in vaccine delivery: facts, challenges and perspectives. Ther Deliv 2019; 10(8): 465-7.
[http://dx.doi.org/10.4155/tde-2019-0042] [PMID: 31462173]
[71]
Brooks NA, Pouniotis DS, Tang CK, Apostolopoulos V, Pietersz GA. Cell-penetrating peptides: application in vaccine delivery. Biochim Biophys Acta 2010; 1805(1): 25-34.
[http://dx.doi.org/10.1016/j.bbcan.2009.09.004] [PMID: 19782720]