Advertisement

Biotechnology Letters

, Volume 41, Issue 11, pp 1283–1298 | Cite as

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides

  • Saba Davoodi
  • Azam BolhassaniEmail author
  • Seyed Mehdi Sadat
  • Shiva Irani
Original Research Paper

Abstract

Objectives

Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system.

Results

Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef60–84, Nef126–144, Vpr34–47, Vpr60–75, Gp16030–53, Gp160308–323 and P248–151 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32 kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293 T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200 nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293 T cells.

Conclusion

The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

Keywords

HIV-1 In silico studies Cell penetrating peptides Vaccine development 

Notes

Supporting information

Supplementary Table 1—Top-ranked CABS-dock models of peptide-HLA I complexes.

Supplementary Table 2—Top-ranked CABS-dock models of peptide-HLA II complexes.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10529_2019_2734_MOESM1_ESM.docx (1.3 mb)
Supplementary file1 (DOCX 1310 kb)
10529_2019_2734_MOESM2_ESM.docx (1.6 mb)
Supplementary file2 (DOCX 1599 kb)

References

  1. Arnon R, Ben-Yedidia T (2003) Old and new vaccine approaches. Int Immunopharmacol 3:1195–1204.  https://doi.org/10.1016/s1567-5769(03)00016-x CrossRefPubMedGoogle Scholar
  2. Bechara C, Sagan S (2013) Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett 587:1693–1702.  https://doi.org/10.1016/j.febslet.2013.04.031 CrossRefPubMedGoogle Scholar
  3. Chin‘Ombe N, Ruhanya V (2015) HIV/AIDS vaccines for Africa: scientific opportunities, challenges and strategies. Pan Afr Med J.  https://doi.org/10.11604/pamj.2015.20.386.4660 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chiozzini C, Toschi E (2015) HIV-1 tat and immune dysregulation in AIDS pathogenesis: a therapeutic target. Curr Drug Targ 17:33–45.  https://doi.org/10.2174/1389450116666150825110658 CrossRefGoogle Scholar
  5. Conner SD, Schmid SL (2003) Regulated portals of entry into the cell. Nature 422:37–44.  https://doi.org/10.1038/nature01451 CrossRefPubMedGoogle Scholar
  6. Degroot A (2003) Mapping cross-clade HIV-1 vaccine epitopes using a bioinformatics approach. Vaccine 21:4486–4504.  https://doi.org/10.1016/s0264-410x(03)00390-6 CrossRefGoogle Scholar
  7. Dubovskii PV, Vassilevski AA, Kozlov SA et al (2015) Latarcins: versatile spider venom peptides. Cell Mol Life Sci 72:4501–4522.  https://doi.org/10.1007/s00018-015-2016-x CrossRefPubMedGoogle Scholar
  8. Eaton P, Quaresma P, Soares C et al (2017) A direct comparison of experimental methods to measure dimensions of synthetic nanoparticles. Ultramicroscopy 182:179–190.  https://doi.org/10.1016/j.ultramic.2017.07.001 CrossRefPubMedGoogle Scholar
  9. Esparza J (2013) What has 30 years of HIV vaccine research taught us? Vaccines 1:513–526.  https://doi.org/10.3390/vaccines1040513 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Esteves A, Parreira R, Venenno T et al (2002) Molecular epidemiology of HIV type 1 infection in portugal: High prevalence of non-B subtypes. AIDS Res Hum Retroviruses 18:313–325.  https://doi.org/10.1089/088922202753519089 CrossRefPubMedGoogle Scholar
  11. Goede AD, Vulto A, Osterhaus A, Gruters R (2015) Understanding HIV infection for the design of a therapeutic vaccine. Part I: epidemiology and pathogenesis of HIV infection. Ann Pharm Françaises 73:87–99.  https://doi.org/10.1016/j.pharma.2014.11.002 CrossRefGoogle Scholar
  12. Gros E, Deshayes S, Morris MC, et al (2006) A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. Biochimica et Biophysica Acta (BBA)—Biomembranes 1758:384–393.  https://doi.org/10.1016/j.bbamem.2006.02.006 CrossRefGoogle Scholar
  13. Guidotti G, Brambilla L, Rossi D (2017) Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol Sci 38:406–424.  https://doi.org/10.1016/j.tips.2017.01.003 CrossRefPubMedGoogle Scholar
  14. Hanna Z, Kay DG, Rebai N et al (1998) Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95:163–175.  https://doi.org/10.1016/s0092-8674(00)81748-1 CrossRefPubMedGoogle Scholar
  15. Herce H, Garcia A, Litt J et al (2009) Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell-penetrating peptides. Biophys J 97:1917–1925.  https://doi.org/10.1016/j.bpj.2009.05.066 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Huang Y-W, Lee H-J, Tolliver LM, Aronstam RS (2015) Delivery of nucleic acids and nanomaterials by cell-penetrating peptides: opportunities and challenges. Biomed Res Int 2015:1–16.  https://doi.org/10.1155/2015/834079 CrossRefGoogle Scholar
  17. Jha D, Mishra R, Gottschalk S et al (2011) CyLoP-1: A novel cysteine-rich cell-penetrating peptide for cytosolic delivery of cargoes. Bioconjug Chem 22:319–328.  https://doi.org/10.1021/bc100045s CrossRefPubMedGoogle Scholar
  18. Kaminchik J, Margalit R, Yaish S et al (1994) Cellular distribution of HIV type 1 Nef protein: Identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix. AIDS Res Hum Retroviruses 10:1003–1010.  https://doi.org/10.1089/aid.1994.10.1003 CrossRefPubMedGoogle Scholar
  19. Karjoo Z, Mccarthy HO, Patel P et al (2013) Systematic engineering of uniform, highly efficient, targeted and shielded viral-mimetic nanoparticles. Small 9:2774–2783.  https://doi.org/10.1002/smll.201300077 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kawamoto S, Takasu M, Miyakawa T et al (2011) Inverted micelle formation of cell-penetrating peptide studied by coarse-grained simulation: importance of attractive force between cell-penetrating peptides and lipid head group. J Chem Phy 134:095103.  https://doi.org/10.1063/1.3555531 CrossRefGoogle Scholar
  21. Kelly J, Beddall MH, Yu D et al (2008) Human macrophages support persistent transcription from unintegrated HIV-1 DNA. Virology 372:300–312.  https://doi.org/10.1016/j.virol.2007.11.007 CrossRefPubMedGoogle Scholar
  22. Khairkhah N, Namvar A, Kardani K, Bolhassani A (2018) Prediction of cross-clade HIV-1 T cell epitopes using immunoinformatics analysis. Proteins 86:1284–1293.  https://doi.org/10.1002/prot.25609 CrossRefPubMedGoogle Scholar
  23. Kunwar P, Hawkins N, Dinges WL et al (2013) Superior Control of HIV-1 Replication by CD8 T cells targeting conserved epitopes: implications for HIV vaccine design. PLoS ONE.  https://doi.org/10.1371/journal.pone.0064405 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kurcinski M, Jamroz M, Blaszczyk M et al (2015) CABS-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site. Nucleic Acids Res.  https://doi.org/10.1093/nar/gkv456 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Laufer S, Restle T (2008) Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems. Curr Pharma Des 14:3637–3655.  https://doi.org/10.2174/138161208786898806 CrossRefGoogle Scholar
  26. Layek B, Lipp L, Singh J (2015) Cell penetrating peptide conjugated chitosan for enhanced delivery of nucleic acid. Int J Mol Sci 16:28912–28930.  https://doi.org/10.3390/ijms161226142 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Leal L, Lucero C, Gatell JM et al (2017) New challenges in therapeutic vaccines against HIV infection. Expert Rev Vaccines 16:587–600.  https://doi.org/10.1080/14760584.2017.1322513 CrossRefPubMedGoogle Scholar
  28. Li C, Shen Z, Li X et al (2012) Protection against SHIV-KB9 infection by combining rDNA and rFPV vaccines based on HIV multiepitope and p24 protein in chinese rhesus macaques. Clin Dev Immunol 2012:1–9.  https://doi.org/10.1155/2012/958404 CrossRefGoogle Scholar
  29. Liu BR, Chen HH, Chan MH et al (2015) Three arginine-rich cell-penetrating peptides facilitate cellular internalization of red-emitting quantum dots. J Nanosci Nanotechnol 15:2067–2078.  https://doi.org/10.1166/jnn.2015.9148 CrossRefPubMedGoogle Scholar
  30. Lo SL, Wang S (2008) An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials 29:2408–2414.  https://doi.org/10.1016/j.biomaterials.2008.01.031 CrossRefPubMedGoogle Scholar
  31. Lundberg P, Langel U (2003) A brief introduction to cell-penetrating peptides. J Mol Recognit 16:227–233.  https://doi.org/10.1002/jmr.630 CrossRefPubMedGoogle Scholar
  32. Mann A, Shukla V, Khanduri R et al (2014) Linear short histidine and cysteine modified arginine peptides constitute a potential class of DNA delivery agents. Mol Pharm 11:683–696.  https://doi.org/10.1021/mp400353n CrossRefPubMedGoogle Scholar
  33. Miller RH, Sarver N (1997) HIV accessory proteins as therapeutic targets. Nat Med 3:389–394.  https://doi.org/10.1038/nm0497-389 CrossRefPubMedGoogle Scholar
  34. Milletti F (2012) Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17:850–860.  https://doi.org/10.1016/j.drudis.2012.03.002 CrossRefPubMedGoogle Scholar
  35. Morris M (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 25:2730–2736.  https://doi.org/10.1093/nar/25.14.2730 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Motevalli F, Bolhassani A, Hesami S, Shahbazi S (2018) Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo. Immunol Lett 194:29–39.  https://doi.org/10.1016/j.imlet.2017.12.005 CrossRefPubMedGoogle Scholar
  37. Perrin H, Canderan G, Sékaly RP, Trautmann L (2010) New approaches to design HIV-1 T-cell vaccines. Curr Opin HIV AIDS 5:368–376.  https://doi.org/10.1097/coh.0b013e32833d2cc0 CrossRefPubMedGoogle Scholar
  38. Ponnappan N, Budagavi DP, Chugh A (2017) CyLoP-1: membrane-active peptide with cell-penetrating and antimicrobial properties. Biochimica et Biophysica Acta (BBA)—Biomembranes 1859:167–176.  https://doi.org/10.1016/j.bbamem.2016.11.002 CrossRefGoogle Scholar
  39. Ponnappan N, Chugh A (2017) Cell-penetrating and cargo-delivery ability of a spider toxin-derived peptide in mammalian cells. Eur J Pharm Biopharm 114:145–153.  https://doi.org/10.1016/j.ejpb.2017.01.012 CrossRefPubMedGoogle Scholar
  40. Pooga M, Langel Ü (2015) Classes of cell-penetrating peptides. Methods Mol Biol 1324:3–28.  https://doi.org/10.1007/978-1-4939-2806-4_1 CrossRefPubMedGoogle Scholar
  41. Puls RL, Emery S (2006) Therapeutic vaccination against HIV: current progress and future possibilities. Clin Sci 110:59–71.  https://doi.org/10.1042/cs20050157 CrossRefPubMedGoogle Scholar
  42. Ragin AD, Morgan RA, Chmielewski J (2002) Cellular import mediated by nuclear localization signal peptide sequences. Chem Biol 9:943–948.  https://doi.org/10.1016/s1074-5521(02)00189-8 CrossRefPubMedGoogle Scholar
  43. Rahmat D, Khan MI, Shahnaz G et al (2012) Synergistic effects of conjugating cell penetrating peptides and thiomers on non-viral transfection efficiency. Biomaterials 33:2321–2326.  https://doi.org/10.1016/j.biomaterials.2011.11.046 CrossRefPubMedGoogle Scholar
  44. Rejman J, Oberle V, Zuhorn IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377:159–169.  https://doi.org/10.1042/bj20031253 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Requejo HIZ (2006) Worldwide molecular epidemiology of HIV. Rev de Saúde Pública 40:331–345.  https://doi.org/10.1590/s0034-89102006000200023 CrossRefGoogle Scholar
  46. Sabouri-Rad S, Oskuee RK, Mahmoodi A et al (2017) The effect of cell penetrating peptides on transfection activity and cytotoxicity of polyallylamine. BioImpacts 7:139–145.  https://doi.org/10.15171/bi.2017.17 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Saleh T, Bolhassani A, Shojaosadati SA, Aghasadeghi MR (2015) MPG-based nanoparticle: an efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7. Vaccine 33:3164–3170.  https://doi.org/10.1016/j.vaccine.2015.05.015 CrossRefPubMedGoogle Scholar
  48. Simeoni F (2003) Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 31:2717–2724.  https://doi.org/10.1093/nar/gkg385 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Temsamani J, Vidal P (2004) The use of cell-penetrating peptides for drug delivery. Drug Discov Today 9:1012–1019.  https://doi.org/10.1016/s1359-6446(04)03279-9 CrossRefPubMedGoogle Scholar
  50. Wei B, Arora V, Foster J et al (2003) In vivo analysis of Nef function. Curr HIV Res 1:41–50.  https://doi.org/10.2174/1570162033352057 CrossRefPubMedGoogle Scholar
  51. Yoo JW, Doshi N, Mitragotri S (2011) Adaptive micro and nanoparticles: temporal control over carrier properties to facilitate drug delivery. Adv Drug Deliv Rev 63:1247–1256.  https://doi.org/10.1016/j.addr.2011.05.004 CrossRefPubMedGoogle Scholar
  52. Zorko M, Langel U (2005) Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev 57:529–545.  https://doi.org/10.1016/j.addr.2004.10.010 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Saba Davoodi
    • 1
  • Azam Bolhassani
    • 2
    Email author
  • Seyed Mehdi Sadat
    • 2
  • Shiva Irani
    • 1
  1. 1.Department of Biology, School of Basic Science, Science and Research BranchIslamic Azad UniversityTehranIran
  2. 2.Department of Hepatitis and AIDSPasteur Institute of IranTehranIran

Personalised recommendations