Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

Document Type : Original Article

Authors

1 Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran , Iran

2 Biotechnology Research Center, Kermanshah University, Kermanshah, Iran

3 Department of Virology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Objective(s):Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine.
Materials and Methods: In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture            medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting.
Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. [AGA1] .
Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. [AGA2] The final concentration of purified protein was 500 µg/ml.

Keywords


1.   Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med 2009; 60:471-484.
2.   Chen YH, Xiao Y, Yu T, Dierich MP. Epitope vaccine: a new strategy against HIV-1. Immunol Today 1999; 20:588-589.
3.   Xiao Y, Lu Y, Chen YH. Epitope-vaccine as a new strategy against HIV-1 mutation. Immunol Lett 2001; 77:3-6.
4.   Liu Z, Xiao Y, Chen YH. Epitope-vaccine strategy against HIV-1: today and tomorrow. Immunobiology 2003; 208:423-428.
5.   Smith SG. The polyepitope approach to DNA vaccination. Curr Opin Mol Ther 1999; 1:10-15.
6.   Baird M, Wilson R, Young L, Williman J, Young S, Wilson M, et al. Bystander help within a polyepitope DNA vaccine improves immune responses to influenza antigens. Scand J Immunol 2004; 60:363-371.
7.   Oyarzun P, Ellis JJ, Boden M, Kobe B. Predivac: CD4+ T-cell epitope prediction for vaccine design that covers 95% of HLA class II DR protein diversity. BMC Bioinformatics 2013; 14:15.
8.   Changela A, Wu X, Yang Y, Zhang B, Zhu J, Nardone GA, et al. Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1. J Virol 2011; 85:2524-2535.
9.   Zhang SM, Song M, Yang TY, Fan R, Liu XD, Zhou PK. HIV-1 Tat impairs cell cycle control by targeting the Tip60, Plk1 and cyclin B1 ternary complex. Cell Cycle 2012; 11:1217-1234.
10. Shortman K, Lahoud MH, Caminschi I. Improving vaccines by targeting antigens to dendritic cells. Exp Mol Med 2009; 41:61-66.
11. Chappell CP, Giltiay NV, Dresch C, Clark EA. Controlling immune responses by targeting antigens to dendritic cell subsets and B cells. Int Immunol 2014; 26:3-11. 
12. Nchinda G, Amadu D, Trumpfheller C, Mizenina O, Uberla K, Steinman RM. Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells. Proc Natl Acad Sci U S A 2010; 107:4281-4286. 
13. Tacken PJ, de Vries IJ, Torensma R, Figdor CG. Dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting. Nat Rev Immunol 2007; 7:790-802.
14. Reis e Sousa C, Hieny S, Scharton-Kersten T, Jankovic D, Charest H, Germain RN, et al. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J Exp Med 1997; 186:1819-1829.
15. Hochrein H, Shortman K, Vremec D, Scott B, Hertzog P, O'Keeffe M. Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets. J Immunol 2001; 166:5448-5455.
16. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S, et al. Differential antigen processing by dendritic cell subsets in vivo. Science 2007; 315:107-111.
17. Boscardin SB, Hafalla JC, Masilamani RF, Kamphorst AO, Zebroski HA, Rai U, et al. Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses. J Exp Med 2006; 203:599-606.
18. Soares H, Waechter H, Glaichenhaus N, Mougneau E, Yagita H, Mizenina O, et al. A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma             by an IL-12-independent but CD70-dependent mechanism in vivo. J Exp Med 2007; 204:1095-1106.
19. Trumpfheller C, Finke JS, Lopez CB, Moran TM, Moltedo B, Soares H, et al. Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine. J Exp Med 2006; 203:607-617.
20. Zheng C, Zhao Z, Li Y, Wang L, Su Z. Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli. Protein Expr Purif 2011; 75:133-137. 
21. Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 1996; 260:289-298.
22. Tamerler CY, Önsan Zİ, Kirdar B. Optimization of starting time and period of induction and inducer concentration in the production of the restriction enzyme EcoRI from recombinant Escherichia coli 294. Turkish J Chemistry  1998; 22:221-226.
23. Sorensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 2005; 4:1.
24. Jafarpour N, Memarnejadian A, Aghasadeghi MR, Kohram F, Aghababa H, Khoramabadi N, et al. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice. Mol Biol Rep 2014; 41:5207-5214. 
25. Kim YS, Seo JH, Cha HJ. Enhancement of heterologous protein expression in Escherichia coli by co-expression of nonspecific DNA-binding stress protein, Dps. Enzyme Microbial Tech 2003; 33:460-465.
26. Hermanson GT. Bioconjugate Techniques. Waltham: Academic Press; 2008.
27. Mahdavi M, Ebtekar M, Azadmanesh K, Khorramkhorshid HR, Rahbarizadeh F, Yazdi MH,            et al. HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model. Acta Virol 2010; 54:131-136.
28. Tegel H, Ottosson J, Hober S. Enhancing the protein production levels in Escherichia coli with a strong promoter. FEBS J 2011; 278:729-739.
29. McBrien DC, Moses V. Effect of isopropylthio-galactoside on induction of the galactose operon by D-fucose in a lactose deletion mutant of Escherichia coli.              
30. Donovan RS, Robinson CW, Glick BR. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J Ind Microbiol 1996; 16:145-154.
31. Bonifaz LC, Bonnyay DP, Charalambous A, Darguste DI, Fujii S, Soares H, et al. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. J Exp Med 2004; 199:815-824.