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

Author(s): Bahareh Rostami, Shiva Irani, Azam Bolhassani*, Reza Ahangari Cohan.

Journal Name: Current HIV Research

Volume 16 , Issue 4 , 2018

Become EABM
Become Reviewer

Graphical Abstract:


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.

Keywords: HIV, Cell-penetrating peptide, Heat shock protein 20, Nef, M918, Transfection.

[1]
HIV/AIDS JUNPo. Global AIDS update 2016 Geneva, Switzerland.
[2]
Mann JK, Ndungu T. HIV-1 vaccine immunogen design strategies. Virol J 2015; 12(1): 3.
[3]
Kirchhoff F. HIV life cycle: overview. Encyclo AIDS 2013; pp. 1-9.
[4]
Geyer M, Fackler OT, Peterlin BM. Structure-function relationships in HIV-1 Nef. EMBO Rep 2001; 2(7): 580-5.
[5]
Milani A, Bolhassani A, Heshmati M. Delivery of HIV-1 Nef linked to heat shock protein 27 using a cationic polymer is more effective than cationic lipid in mammalian cells. Bratisl Lek Listy 2017; 118(6): 334-8.
[6]
Gómez CE, Nájera JL, Jiménez V, et al. Generation and immunogenicity of novel HIV/AIDS vaccine candidates targeting HIV-1 Env/Gag-Pol-Nef antigens of clade C. Vaccine 2007; 25(11): 1969-92.
[7]
García F, Bernaldo de Quirós JC, Gómez CE, et al. Safety and immunogenicity of a modified pox vector-based HIV/AIDS vaccine candidate expressing Env, Gag, Pol and Nef proteins of HIV-1 subtype B (MVA-B) in healthy HIV-1-uninfected volunteers: A phase I clinical trial (RISVAC02). Vaccine 2011; 29(46): 8309-16.
[8]
Safrit JT, Fast PE, Gieber L, Kuipers H, Dean HJ, Koff WC. Status of vaccine research and development of vaccines for HIV-1. Vaccine 2016; 34(26): 2921-5.
[9]
Perdiguero B, Gómez CE, Cepeda V, et al. Virological and immunological characterization of novel NYVAC-based HIV/AIDS vaccine candidates expressing clade C trimeric soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as virus-like particles. J Virol 2015; 89: 970-88.
[10]
Hopkins KL, Laher F, Otwombe K, et al. Predictors of HVTN 503 MRK-AD5 HIV-1 gag/pol/nef vaccine induced immune responses. PLoS One 2014; 9(8): e103446.
[11]
Lema D, Garcia A, De Sanctis JB. HIV vaccines: a brief overview. Scand J Immunol 2014; 80(1): 1-11.
[12]
Basirnejad M, Bolhassani A, Sadat SM. The distinct role of small heat shock protein 20 on HCV NS3 expression in HEK-293T cell line. Avicenna J Med Biotechnol 2018; 10(3): 152-7.
[13]
Thorén PE, Persson D, Karlsson M, Nordén B. The antennapedia peptide penetratin translocates across lipid bilayers–the first direct observation. FEBS Lett 2000; 482(3): 265-8.
[14]
Albrizio S, Giusti L, D’Errico G, et al. Driving forces in the delivery of penetratin conjugated G protein fragment. J Med Chem 2007; 50(7): 1458-64.
[15]
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.
[16]
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): E185.
[17]
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.
[18]
Vivès E, Schmidt J, Pèlegrin A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochimica et Biophysica Acta (BBA). Rev Can 2008; 1786(2): 126-38.
[19]
Madani F, Lindberg S, Langel Ü, Futaki S, Gräslund A. Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011; 2011: 414729.
[20]
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.
[21]
Bolhassani A, Jafarzade BS, Mardani G. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides. Peptides 2017; 87: 50-63.
[22]
Tashima T. Intelligent substance delivery into cells using cell-penetrating peptides. Bioorg Med Chem Lett 2017; 27(2): 121-30.
[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: 12538.
[24]
Säälik P, Elmquist A, Hansen M, et al. Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug Chem 2004; 15(6): 1246-53.
[25]
Jafari S, Maleki Dizaj S, Adibkia K. Cell-penetrating peptides and their analogues as novel nanocarriers for drug delivery. Bioimpacts 2015; 5(2): 103-11.
[26]
Ngwa VM, Axford DS, Healey AN, Nowak SJ, Chrestensen CA, McMurry JL. A versatile cell-penetrating peptide-adaptor system for efficient delivery of molecular cargos to subcellular destinations. PLoS One 2017; 12(5): e0178648.
[27]
Vasconcelos L, Pärn K, Langel U. Therapeutic potential of cell-penetrating peptides. Ther Deliv 2013; 4(5): 573-91.
[28]
Chugh A, Eudes F, Shim YS. Cell penetrating peptides: Nano-carrier for macromolecule delivery in living cells. IUBMB Life 2010; 62(3): 183-93.
[29]
El-Andaloussi S, Johansson HJ, Holm T, Langel U. A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids. Mol Ther 2007; 15: 1820-6.
[30]
Jarver P, Fernaeus S, El-Andaloussi S, Tjornhammar ML, Langel U. Co-transduction of sleeping beauty transposase and donor plasmid via a cell-penetrating peptide: a simple one step method. Int J Pept Res Ther 2008; 14: 58-63.
[31]
Milani A, Bolhassani A, Shahbazi S, Motevalli F, Sadat SM, Soleymani S. Small heat shock protein 27: An effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity. Immunol Lett 2017; 191: 16-22.
[32]
Javanzad S, Bolhassani A, Doustdari F, Hashemi M, Movafagh A. Reverse staining method of polyacrylamide gels by imidazole-zinc salts for detection and purification of L1 capsid protein in E.coli J Paramed Sci 2013; 4(2): 33-7.
[33]
Motevalli F, Bolhassani A, Hesami S, Shahbazi S. Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo. Immunol Lett 2018; 194: 29-39.
[34]
Ajbani S. HIV vaccine development: current scenario and future prospects. J AIDS Clin Res 2016; 7(11): 626.
[35]
Lin L, Li HS, Pauza CD, Bukrinsky M, Richard YZ. Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions. Cell Res 2005; 15(11-12): 923.
[36]
Sugden SM, Bego MG, Pham TN, Cohen EA. Remodeling of the host cell plasma membrane by HIV-1 Nef and Vpu: A strategy to ensure viral fitness and persistence. Viruses 2016; 8(3): 67.
[37]
Segal BH, Wang X-Y, Dennis CG, et al. Heat shock proteins as vaccine adjuvants in infections and cancer. Drug Discov Today 2006; 11(11-12): 534-40.
[38]
McNulty S, Colaco CA, Blandford LE, Bailey CR, Baschieri S, Todryk S. Heat-shock proteins as dendritic cell-targeting vaccines-getting warmer. Immunology 2013; 139(4): 407-15.
[39]
Fan GC, Chu G, Kranias EG. Hsp20 and its cardioprotection. Trends Cardiovasc Med 2005; 15(4): 138-41.
[40]
Montalvo-Alvarez AM, Folgueira C, Carrion J, Monzote-Fidalgo L, Canavate C, Requena JM. The Leishmania HSP20 is antigenic during natural infections, but, as DNA vaccine, it does not protect BALB/c mice against experimental L. amazonensis infection. J Biomed Biotechnol 2008; 695432.
[41]
Brown WC, Ruef BJ, Norimine J, et al. A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4+ T lymphocyte responses in B.bovis-immune cattle. Mol Biochem Parasitol 2001; 118(1): 97-109.
[42]
Norimine J, Mosqueda J, Palmer GH, Lewin HA, Brown WC. Conservation of babesia bovis small heat shock protein (Hsp20) among strains and definition of T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes. Infect Immun 2004; 72(2): 1096-106.
[43]
Ortiz JMJ, Zajac MPDM, Zanetti FA, et al. Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins. Vaccine 2014; 32(36): 4625-32.
[44]
Milani A, Bolhassani A, Shahbazi S, Motevalli F, Sadat SM, Soleymani S. Small heat shock protein 27: an effective adjuvant for enhancement of HIV-1 Nef antigen-specific immunity. Immunol Lett 2017; 191: 16-22.
[45]
Jafarzade BS, Sadat SM, Yaghobi R, Bolhassani A. Improving the potency of DNA vaccine encoding HIV-1 Nef antigen using two endogenous adjuvants in mouse model. Bratisl Lek Listy 2017; 118(9): 564-9.
[46]
Guo Z, Peng H, Kang J, Sun D. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications (Review). Biomed Rep 2016; 4(5): 528-34.
[47]
Li H, Tsui TY, Ma W. Intracellular delivery of molecular cargo using cell-penetrating peptides and the combination strategies. Int J Mol Sci 2015; 16(8): 19518-36.
[48]
Cui Z, Patel J, Tuzova M, et al. Strong T cell type-1 immune responses to HIV-1 Tat (1–72) protein-coated nanoparticles. Vaccine 2004; 22: 2631-40.
[49]
Torchilin V. Intracellular delivery of protein and peptide therapeutics. Drug Discov Today Technol 2009; 5: e95-e103.
[50]
Heitz F, Morris MC, Divita G. Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br J Pharmacol 2009; 157: 195-206.
[51]
Munyendo WLL, Lv H, Benza-Ingoula H, Baraza LD, Zhou L. Cell penetrating peptides in the delivery of biopharmaceuticals. Biomolecules 2012; 2: 187-202.
[52]
Kadkhodayan S, Jafarzade BS, Sadat SM, Motevalli F, Agi E, Bolhassani A. Combination of cell penetrating peptides and heterologous DNA prime/protein boost strategy enhances immune responses against HIV-1 Nef antigen in BALB/c mouse model. Immunol Lett 2017; 188: 38-45.
[53]
Kadkhodayan S, Bolhassani A, Sadat SM, Irani S, Fotouhi F. The efficiency of Tat cell penetrating peptide for intracellular uptake of HIV-1 Nef expressed in E. coli and mammalian cell. Curr Drug Deliv 2017; 14(4): 536-42.
[54]
Sawant RR, Patel NR, Torchilin VP. Therapeutic delivery using cell-penetrating peptides. Eur J Nanomed 2013; 5(3): 141-58.
[55]
Ruczynski EJ, Wierzbicki PM, Kogut-Wierzbicka M, Mucha P, Siedlecka-Kroplewska K, Rekowski P. Cell-penetrating peptides as a promising tool for delivery of various molecules into the cells. Folia Histochem Cytobiol 2014; 52(4): 257-69.
[56]
Holm T, Räägel H, Andaloussi SEL, et al. Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity. Biochim Biophys Acta 2011; 1808: 1544-51.
[57]
Eiríksdóttir E, Konate K, Langel U, Divita G, Deshayes S. Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochim Biophys Acta 2010; 1798: 1119-28.
[58]
Deshayes S, Heitz A, Morris MC, Charnet P, Divita G, Heitz F. Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis. Biochemistry 2004; 43: 1449-57.
[59]
Konate K, Crombez L, Deshayes S, et al. Insight into the cellular uptake mechanism of a secondary amphipathic cell penetrating peptide for siRNA delivery. Biochemistry 2010; 49(16): 3393-402.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 16
ISSUE: 4
Year: 2018
Page: [280 - 287]
Pages: 8
DOI: 10.2174/1570162X17666181206111859

Article Metrics

PDF: 20
HTML: 6