Volume 7, Issue 4 (10-2019)                   JoMMID 2019, 7(4): 107-115 | Back to browse issues page


XML Print


Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (4263 Views)
Introduction: The Nef accessory protein is an attractive antigenic candidate in the development of HIV-1 DNA- or protein-based vaccines. The most crucial disadvantage of DNA and protein-based vaccines is their low immunogenicity, which can be improved by cell-penetrating peptides (CPPs) as effective carrier molecules. Methods: In this study, the HIV-1 Nef protein was generated in the Escherichia coli expression system for in vitro delivery using a novel CPP, Latarcin 1 peptide, in a non-covalent manner. Also, the Histidine-rich nona-arginine peptide was utilized to transfer the HIV-1 Nef gene. The size, morphology, and zeta potential of the complexes were evaluated by scanning electron microscopy (SEM) and Zetasizer. The efficiency of cell transfection was studied using a fluorescence microscopy and flow cytometry for the DNA/CPP complexes and western blot analysis for the protein/CPP complexes. Results: The Nef protein generated in the BL21 strain migrated as a dominant band of ~30 kDa in SDS-PAGE. The SEM data confirmed the formation of stable complexes with a size below 200 nm. MTT assay demonstrated that the complexes did not represent any considerable cytotoxic effect compared to untreated HEK-293T cells. The results of fluorescence microscopy, flow cytometry, and western blotting revealed that the Nef DNA and protein constructs could be significantly transfected into HEK-293T cell line using these CPPs. Conclusion: These data suggest that the Histidine-rich nona-arginine peptide and Latarcin 1 peptide as CPPs can be considered as a promising approach in the development of the HIV-1 vaccine for gene or protein delivery. 
Full-Text [PDF 893 kb]   (1191 Downloads)    
Type of Study: Original article | Subject: Other
Received: 2019/07/27 | Accepted: 2019/10/14 | Published: 2020/03/12

References
1. 1. Alves BM, Siqueira JD, Prellwitz IM, Botelho OM, Da Hora VP, Sanabani S, et al. Estimating HIV-1 genetic diversity in Brazil through next-generation sequencing. Front Microbiol. 2019; 10. [DOI:10.3389/fmicb.2019.00749]
2. Seitz R. Human Immunodeficiency Virus (HIV). Transfus Med Hemother. 2016; 43 (3): 203-22. [DOI:10.1159/000445852]
3. Li G, Piampongsant S, Faria NR, Voet A, Pineda-Peña A-C, Khouri R, et al. An integrated map of HIV genome-wide variation from a population perspective. Retrovirology. 2015; 12 (1). [DOI:10.1186/s12977-015-0148-6]
4. Khairkhah N, Namvar A, Kardani K, Bolhassani A. Prediction of cross‐clade HIV‐1 T‐cell epitopes using immunoinformatics analysis. Proteins: Structure, Function, and Bioinformatics. 2018; 86 (12): 1284-93. [DOI:10.1002/prot.25609]
5. Bråve A, Gudmundsdotter L, Gasteiger G, Hallermalm K, Kastenmuller W, Rollman E, et al. Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology. Infect Agent Cancer. 2007; 2 (1). [DOI:10.1186/1750-9378-2-14]
6. Jafarzade B, Sadat S, 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 (09): 564-569. [DOI:10.4149/BLL_2017_108]
7. Quaranta MG, Mattioli B, Giordani L, Viora M. Immunoregulatory effects of HIV-1 Nef protein. BioFactors. 2009; 35 (2): 169-174. [DOI:10.1002/biof.28]
8. 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. [DOI:10.3389/fmicb.2017.01022]
9. 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. [DOI:10.1016/j.vaccine.2018.03.042]
10. Lema D, Garcia A, De Sanctis J. HIV vaccines: A brief overview. Scand J Immunol. 2014; 80 (1): 1-11. [DOI:10.1111/sji.12184]
11. Kadkhodayan S, Jafarzade B, Sadat S, 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. [DOI:10.1016/j.imlet.2017.06.003]
12. Alhakamy NA, Nigatu AS, Berkland CJ, Ramsey JD. Noncovalently associated cell-penetrating peptides for gene delivery applications. Ther Deliv. 2013; 4 (6): 741-57. [DOI:10.4155/tde.13.44]
13. 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. [DOI:10.1016/j.peptides.2014.04.015]
14. 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. [DOI:10.2174/1570162X17666181206111859]
15. Liu BR, Huang Y, 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. [DOI:10.1016/j.biomaterials.2011.01.041]
16. 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. [DOI:10.1016/j.ejpb.2017.01.012]
17. 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. [DOI:10.1016/j.imlet.2017.09.005]
18. Javanzad S, Bolhassani A, Doustdari F, Hashemi M, Movafagh A. Reverse staining method of polyacrylamide gels by imidazole-zinc salts for. J Paramed Sci. 2013; 4 (2).
19. Cosma A, Nagaraj R, Bühler S, Hinkula J, Busch D, Sutter G, et al. Therapeutic vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine. 2003; 22 (1): 21-9. [DOI:10.1016/S0264-410X(03)00538-3]
20. Billaut-Mulot O, Idziorek T, Ban E, Kremer L, Dupré L, Loyens M, et al. Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine. Vaccine. 2000; 19 (1): 95-102. [DOI:10.1016/S0264-410X(00)00157-2]
21. Asakura Y, Hamajima K, Fukushima J, Mohri H, Okubo T, Okuda K. Induction of HIV‐1 Nef‐specific cytotoxic T lymphocytes by Nef‐expressing DNA vaccine. Am J Hematol. 1996; 53 (2): 116-117. https://doi.org/10.1002/(SICI)1096-8652(199610)53:2<116::AID-AJH9>3.0.CO;2-2 [DOI:10.1002/(SICI)1096-8652(199610)53:23.0.CO;2-2]
22. Harrer E, Bäuerle M, Ferstl B, Chaplin P, Petzold B, Mateo L, et al. Therapeutic vaccination of HIV-1-infected patients on HAART with a recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antivir Ther. 2005; 10 (2): 285-300.
23. Madani F, Lindberg S, Langel Ü, Futaki S, Gräslund A. Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys. 2011; 2011: 1-10. [DOI:10.1155/2011/414729]
24. Rathnayake PVGM, Gunathunge BGCM, Wimalasiri PN, Karunaratne DN, Ranatunga RJKU. Trends in the binding of cell penetrating peptides to siRNA: A molecular docking study. J Biophys. 2017; 2017: 1-12. [DOI:10.1155/2017/1059216]
25. Keller AA, Mussbach F, Breitling R, Hemmerich P, Schaefer B, Lorkowski S, et al. Relationships between cargo, cell penetrating peptides and cell type for uptake of non-covalent complexes into live cells. Pharmaceuticals. 2013; 6 (2): 184-203. [DOI:10.3390/ph6020184]
26. Durzynska J, Przysiecka ucja, Nawrot R, Barylski J, Nowicki G, Warowicka A, et al. Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine. J Pharmacol Exp Ther. 2015; 354 (1): 32-42. [DOI:10.1124/jpet.115.223305]
27. Chen Y-J, Liu BR, Dai Y-H, Lee C-Y, Chan M-H, Chen H-H, et al. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene. 2012; 493 (2): 201-10. [DOI:10.1016/j.gene.2011.11.060]
28. 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. [DOI:10.1016/j.gene.2012.05.053]
29. 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. Marine Biotechnol. 2013; 15 (5): 584-95. [DOI:10.1007/s10126-013-9509-0]
30. Mandraccia L, Slavin G. Cell membrane. Hauppauge: Nova Science Publishers, Inc; 2013.
31. Laufer S, 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. [DOI:10.2174/138161208786898806]
32. Dubovskii P, Vassilevski A, Kozlov S, Feofanov A, Grishin E, Efremov R. Latarcins: versatile spider venom peptides. Cell Mol Life Sci. 2015; 72 (23): 4501-22. [DOI:10.1007/s00018-015-2016-x]

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.