Evaluation of immune response to recombinant Bacillus anthracis LFD1-PA4 chimeric protein

Document Type: Full paper (Original article)

Authors

1 Ph.D. Student in Nano Biotechnology, Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran

2 Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran

3 MSc Student in Cellular and Molecular Biology, Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran

Abstract

Background: Anthrax is a particularly dangerous infectious disease that affects humans and livestock. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. Domains 4 and 1 of the protective antigen (PA) and lethal factor (LF), respectively, have very high antigenic properties. Aims: In this experimental study, the pET28a-lfD1-pa4 expression vector was designed, constructed and transferred into E. coli BL21 (DE3) plysS. Methods: For this purpose, pa4 gene was amplified by polymerase chain reaction (PCR) and cloned in a pGEM T-easy vector. The pGEM-pa4 and pGEM-lfD1 were digested by XbaI and HindIII enzymes. The ligation reaction was performed by ligase T4 enzyme and the gene cassette, lfD1-pa4, was subcloned in pET28a and transferred to E. coli BL21 (DE3) PlysS. Expression and purification of chimeric proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. The chimera LFD1-PA4 and mixed LFD1+PA4 proteins were injected four times into mice and antibody production was relativity evaluated by enzyme-linked immunosorbent assay (ELISA) test. Results: The results showed that both chimeric and mixed proteins are immunogenic, but LFD1-PA4 has a higher potential to stimulate mice immune system. Conclusion: LFD1-PA4 chimeric protein induced a higher immune response than LFD1+PA4 mixed protein and elicited antibody responses to LF and edema factor (EF), therefore, it holds promise to be a more effective trivalent vaccine candidate to use in anthrax prevention.

Keywords


Ahmadi, AH; Honari, H and Minaei, ME (2015). Cloning, fusion, and expression of domain a-1 protective antigen (PA20) of Bacillus anthracis and N-terminal ipaD gene of Shigella in E. coli. Qom Univ. Med. Sci. J., 9: 20-29.

Aillie, LW; Huwar, TB; Moore, S; Mellado-Sanchez, G; Rodriguez, L; Neeson, BN; Flick-Smith, HC; Jenner, DC; Atkins, HS and Ingram, RJ (2010). An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor. Vaccine. 28: 6740-6748.

Albrecht, MT; Li, H; Williamson, ED; LeButt, CS; Flick-Smith, HC; Quinn, CP; Westra, H; Galloway, D; Mateczun, A and Goldman, S (2007). Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax. Infect. Immun., 75: 5425-5433.

Aulinger, BA; Roehrl, MH; Mekalanos, JJ; Collier, RJ and Wang, JY (2005). Combining anthrax vaccine and therapy: a dominant-negative inhibitor of anthrax toxin is also a potent and safe immunogen for vaccines. Infect. Immun., 73: 3408-3414.

Baillie, L; Townend, T; Walker, N; Eriksson, U and Williamson, D (2004). Characterization of the human immune response to the UK anthrax vaccine. FEMS Immunol. Med. Microbiol., 42: 267-270.

Bellanti, JA; Lin, FYC; Chu, C; Shiloach, J; Leppla, SH; Benavides, GA; Karpas, A; Moayeri, M; Guo, C and Robbins, JB (2012). Phase 1 study of a recombinant mutant protective antigen of Bacillus anthracis. Clin. Vaccine Immunol., 19: 140-145.

Bergman, NH (2010). Bacillus anthracis and anthrax. 1st Edn., Amazon, USA, Wiley-Blackwell. P: 475s.

Bollag, D; Rozycki, M and Edelstein, S (1996). Protein methods. 2nd Edn., ISBN: 978-0-471-11837-4, Wiley-Blackwell. P: 432s.

Bouzianas, DG (2010). Current and future medical approaches to combat the anthrax threat. J. Med. Chem., 53: 4305-4331.

Bradley, KA; Mogridge, J; Mourez, M; Collier, RJ and Young, JA (2001). Identification of the cellular receptor for anthrax toxin. Nature. 414: 225.

Bragg, TS and Robertson, DL (1989). Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis. Gene. 81: 45-54.

Brown, BK; Cox, J; Gillis, A; VanCott, TC; Marovich, M; Milazzo, M; Antonille, TS; Wieczorek, L; McKee Jr, KT and Metcalfe, K (2010). Phase I study of safety and immunogenicity of an Escherichia coli-derived recombinant protective antigen (rPA) vaccine to prevent anthrax in adults. PLoS One. 5: e13849.

Campbell, JD; Clement, KH; Wasserman, SA; Donegan, S; Chrisley, L and Kotloff, KL (2007). Safety, reactogenicity, and immunogenicity of a recombinant protective antigen anthrax vaccine given to healthy adults. Hum. Vaccin. 3: 205-211.

Crowe, SR; Garman, L; Engler, RJ; Farris, AD; Ballard, JD; Harley, JB and James, JA (2011). Anthrax vaccination induced anti-lethal factor IgG: fine specificity and neutralizing capacity. Vaccine. 29: 3670-3678.

Galloway, D; Liner, A; Legutki, J; Mateczun, A; Barnewall, R and Estep, J (2004). Genetic immunization against anthrax. Vaccine. 22: 1604-1608.

Gilligan, PH (2002). Therapeutic challenges posed by bacterial bioterrorism threats. Curr. Opin. Microbiol., 5: 489-495.

Gilligan, PH (2004). The development of antimicrobials and vaccines against bacterial bioterrorism agents--where are we? Drug Discov. Today. 9: 205-206.

Gordon, VM; Rehemtulla, A and Leppla, SH (1997). A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen. Infect. Immun., 65: 3370-3375.

Gorse, GJ; Keitel, W; Keyserling, H; Taylor, DN; Lock, M; Alves, K; Kenner, J; Deans, L and Gurwith, M (2006). Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: a randomized, double-blinded, controlled, multicenter trial. Vaccine. 24: 5950-5959.

Hepburn, MJ; Dyson, EH; Simpson, AJ; Brenneman, KE; Bailey, N; Wilkinson, L; Hornby, R; Mateczun, AJ; Bell, MG and Baillie, LW (2007). Immune response to two different dosing schedules of the anthrax vaccine precipitated (AVP) vaccine. Vaccine. 25: 6089-6097.

Honari, H; Mehrazin, H; Saadati, M and Minaei, ME (2014). Production of polycolonal antibody against domain 2-4 of protective antigen of Bacillus anthracis in laboratory animals. J. Shahrekord Univ. Med. Sci., 15: 35-43.

Hwang, BY; Varadarajan, N; Li, H; Rodriguez, S; Iverson, BL and Georgiou, G (2007). Substrate specificity of the Escherichia coli outer membrane protease OmpP. J. Bacteriol., 189: 522-530.

Joseph, S and David, WR (2001). Molecular cloning: a laboratory manual. 3rd Edn., New York, Cold Spring Harbor Laboratory Press. P: 978.

Kintzer, AF; Thoren, KL; Sterling, HJ; Dong, KC; Feld, GK; Tang, II; Zhang, TT; Williams, ER; Berger, JM and Krantz, BA (2009). The protective antigen component of anthrax toxin forms functional octameric complexes. J. Mol. Biol., 392: 614-629.

Klimpel, KR; Molloy, SS; Thomas, G and Leppla, SH (1992). Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc. Natl. Acad. Sci. U.S.A., 89: 10277-10281.

Knight, J (2001). Bioweapons: delivering death in the mail. Nature. 414: 837.

Koehler, TM (2009). Bacillus anthracis physiology and genetics. Mol. Aspects Med., 30: 386-396.

Lacy, DB; Mourez, M; Fouassier, A and Collier, RJ (2002). Mapping the anthrax protective antigen binding site on the lethal and edema factors. J. Biol. Chem., 277: 3006-3010.

Little, S; Leppla, S and Friedlander, A (1990). Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin. Infect. Immun., 58: 1606-1613.

Liu, S; Crown, D; Miller-Randolph, S; Moayeri, M; Wang, H; Hu, H; Morley, T and Leppla, SH (2009a). Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc. Natl. Acad. Sci. U.S.A., 106: 12424-12429.

Liu, TH; Oscherwitz, J; Schnepp, B; Jacobs, J; Yu, F; Cease, KB and Johnson, PR (2009b). Genetic vaccines for anthrax based on recombinant adeno-associated virus vectors. Mol. Ther., 17: 373-379.

Madanchi, H; Honari, H; Safaei, S and Sayadmanesh, A (2012). Fusion of cholera toxin B subunit (ctxB) with Shigella dysenteriae type I toxin B subunit (stxB), cloning and expression that in E. coli. J. Fasa Univ. Med. Sci., 2: 135-141.

McComb, RC and Martchenko, M (2016). Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen—The first step toward a rationally designed anthrax vaccine. Vaccine. 34: 13-19.

Nguyen, ML; Crowe, SR; Kurella, S; Teryzan, S; Cao, B; Ballard, JD; James, JA and Farris, AD (2009). Sequential B-cell epitopes of Bacillus anthracis lethal factor bind lethal toxin-neutralizing antibodies. Infect. Immun., 77: 162-169.

Okinaka, R; Cloud, K; Hampton, O; Hoffmaster, A; Hill, K; Keim, P; Koehler, T; Lamke, G; Kumano, S and Manter, D (1999). Sequence, assembly and analysis of pX01 and pX02. J. Appl. Microbiol., 87: 261-262.

Pannifer, AD; Wong, TY; Schwarzenbacher, R; Renatus, M; Petosa, C; Bienkowska, J; Lacy, DB; Collier, RJ; Park, S and Leppla, SH (2001). Crystal structure of the anthrax lethal factor. Nature. 414: 229.

Pezard, C; Weber, M; Sirard, JC; Berche, P and Mock, M (1995). Protective immunity induced by Bacillus anthracis toxin-deficient strains. Infect. Immun., 63: 1369-1372.

Price, BM; Liner, AL; Park, S; Leppla, SH; Mateczun, A and Galloway, DR (2001). Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect. Immun., 69: 4509-4515.

Ranjbar, R; Dallal, MS; Pourshafie, M; Aslani, M and Majdzadeh, R (2004). Serogroup distribution of Shigella in Tehran. Iran. J. Public Health. 33: 32-35.

Ranjbar, R; Haghi-Ashtiani, M; Jafari, NJ and Abedini, M (2009). The prevalence and antimicrobial susceptibility of bacterial uropathogens isolated from pediatric patients. Iran. J. Public Health. 38: 134-138.

Reed, MD; Wilder, JA; Mega, WM; Hutt, JA; Kuehl, PJ; Valderas, MW; Chew, LL; Liang, BC and Squires, CH (2015). Immunization with a recombinant, pseudomonas
fluorescens-expressed, mutant form of Bacillus anthracis- derived protective antigen protects rabbits from anthrax infection. PLoS One. 10: e0130952.

Rezaee, M; Honari, H and Kooshk, MRA (2014). Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody. Mol. Biol. Rep., 41: 2445-2452.

Saadati, M; Heiat, M; Nazarian, S; Barati, B; Honari, H; Doroudian, M; Hesaraki, M; Malaei, F; Eghtedardoust, M and Rahbar, M (2010). Cloning and expression of N-terminal region of IpaD from Shigella dysenteriae in E. coli. J. Paramed. Sci., 1: 12-17.

Schmidt, TR; Scott, EJ and Dyer, DW (2011). Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group. BMC Genomics. 12: 430.

Schneemann, A and Manchester, M (2009). Anti-toxin antibodies in prophylaxis and treatment of inhalation anthrax. Future Microbiol., 4: 35-43.

Young, JA and Collier, RJ (2007). Anthrax toxin: receptor binding, internalization, pore formation, and translocation. Annu. Rev. Biochem., 76: 243-265.