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An adenoviral vector-based mucosal vaccine is effective in protection against botulism

Gene Therapy volume 16, pages 367–375 (2009)Cite this article

Abstract

A replication-incompetent adenoviral vector encoding the heavy chain C-fragment (HC50) of botulinum neurotoxin type C (BoNT/C) was evaluated as a mucosal vaccine against botulism in a mouse model. Single intranasal inoculation of the adenoviral vector elicited a high level of HC50-specific IgG, IgG1 and IgG2a in sera and IgA in mucosal secretions as early as 2 weeks after vaccination. The antigen-specific serum antibodies were maintained at a high level at least until the 27th week. Immune sera showed high potency in neutralizing BoNT/C as indicated by in vitro toxin neutralization assay. The mice receiving single dose of 2 × 107 p.f.u. (plaque-forming unit) of adenoviral vector were completely protected against challenge with up to 104 × MLD50 of BoNT/C. The protective immunity showed vaccine dose dependence from 105 to 2 × 107 p.f.u. of adenoviral vector. In addition, animals receiving single intranasal dose of 2 × 107 p.f.u. adenoviral vector could be protected against 100 × MLD50 27 weeks after vaccination. Animals with preexisting immunity to adenovirus could also be vaccinated intranasally and protected against lethal challenge with BoNT/C. These results suggest that the adenoviral vector is a highly effective gene-based mucosal vaccine against botulism.

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References

  1. Villar RG, Elliott SP, Davenport KM . Botulism: the many faces of botulinum toxin and its potential for bioterrorism. Infect Dis Clin North Am 2006; 20: 313–327.

    Article  PubMed  Google Scholar 

  2. Atlas RM . Bioterriorism: from threat to reality. Annu Rev Microbiol 2002; 56: 167–185.

    Article  CAS  PubMed  Google Scholar 

  3. Montgomery VA, Makuch RS, Brown JE, Hack DC . The immunogenicity in humans of a botulinum type F vaccine. Vaccine 1999; 18: 728–735.

    Article  CAS  PubMed  Google Scholar 

  4. Takeda M, Kasai H, Torii Y, Mukamoto M, Kohda T, Tsukamoto K et al. Protective effect of botulinum C/D mosaic toxoid against avian botulism. J Vet Med Sci 2006; 68: 325–330.

    Article  CAS  PubMed  Google Scholar 

  5. Bennett AM, Perkins SD, Holley JL . DNA vaccination protects against botulinum neurotoxin type F. Vaccine 2003; 21: 3110–3117.

    Article  CAS  PubMed  Google Scholar 

  6. Segelke B, Knapp M, Kadkhodayan S, Balhorn R, Rupp B . Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity. Proc Natl Acad Sci USA 2004; 101: 6888–6893.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Woodward LA, Arimitsu H, Hirst R, Oguma K . Expression of HC subunits from Clostridium botulinum types C and D and their evaluation as candidate vaccine antigens in mice. Infect Immun 2003; 71: 2941–2944.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Byrne MP, Smith LA . Development of vaccines for prevention of botulism. Biochimie 2000; 82: 955–966.

    Article  CAS  PubMed  Google Scholar 

  9. Oshima M, Hayakari M, Middlebrook JL, Atassi MZ . Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855–1296) of the toxin. Mol Immunol 1997; 34: 1031–1040.

    Article  CAS  PubMed  Google Scholar 

  10. Webb RP, Smith TJ, Wright PM, Montgomery VA, Meagher MM, Smith LA . Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins. Vaccine 2007; 25: 4273–4282.

    Article  CAS  PubMed  Google Scholar 

  11. Byrne MP, Smith TJ, Montgomery VA, Smith LA . Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate. Infect Immun 1998; 66: 4817–4822.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Atassi MZ, Oshima M . Structure, activity, and immune (T and B cell) recognition of botulinum neurotoxins. Crit Rev Immunol 1999; 19: 219–260.

    CAS  PubMed  Google Scholar 

  13. Ravichandran E, Al-Saleem FH, Ancharski DM, Elias MD, Singh AK, Shamim M et al. Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route. Infect Immun 2007; 75: 3043–3054.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Smith LA, Rusnak JM . Botulinum neurotoxin vaccines: past, present, and future. Crit Rev Immunol 2007; 27: 303–318.

    Article  CAS  PubMed  Google Scholar 

  15. Baldwin MR, Tepp WH, Pier CL, Bradshaw M, Ho M, Wilson BA et al. Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E. Infect Immun 2005; 73: 6998–7005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Smith LA, Jensen MJ, Montgomery VA, Brown DR, Ahmed SA, Smith TJ . Roads from vaccines to therapies. Mov Disord 2004; 19: S48–S52.

    Article  PubMed  Google Scholar 

  17. Byrne MP, Titball RW, Holley J, Smith LA . Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris. Protein Expr Purif 2000; 18: 327–337.

    Article  CAS  PubMed  Google Scholar 

  18. Boles J, West M, Montgomery V, Tammariello R, Pitt ML, Gibbs P et al. Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (HC)) immune response and protection in the rhesus monkey. Toxicon 2006; 47: 877–884.

    Article  CAS  PubMed  Google Scholar 

  19. Holley JL, Elmore M, Mauchline M, Minton N, Titball RW . Cloning, expression and evaluation of a recombinant sub-unit vaccine against Clostridium botulinum type F toxin. Vaccine 2000; 19: 288–297.

    Article  CAS  PubMed  Google Scholar 

  20. Lee JS, Pushko P, Parker MD, Dertzbaugh MT, Smith LA, Smith JF . Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system. Infect Immun 2001; 69: 5709–5715.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bangari DS, Mittal SK . Development of nonhuman adenoviruses as vaccine vectors. Vaccine 2006; 24: 849–862.

    Article  CAS  PubMed  Google Scholar 

  22. Tatsis N, Ertl HC . Adenoviruses as vaccine vectors. Mol Ther 2004; 10: 616–629.

    Article  CAS  PubMed  Google Scholar 

  23. Babiuk LA, Tikoo SK . Adenoviruses as vectors for delivering vaccines to mucosal surfaces. J Biotechnol 2000; 83: 105–113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xu Q, Arevalo MT, Pichichero ME, Zeng M . A new complementing cell line for replication-incompetent E1-deleted adenovirus propagation. Cytotechnology 2006; 51: 133–140.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hoelscher MA, Garg S, Bangari DS, Belser JA, Lu X, Stephenson I et al. Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 2006; 367: 475–481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kobinger GP, Figueredo JM, Rowe T, Zhi Y, Gao G, Sanmiguel JC et al. Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques. Vaccine 2007; 25: 5220–5231.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shu Y, Winfrey S, Yang ZY, Xu L, Rao SS, Srivastava I et al. Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs. Vaccine 2007; 25: 1398–1408.

    Article  CAS  PubMed  Google Scholar 

  28. Hu RL, Liu Y, Zhang SF, Zhang F, Fooks AR . Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies. Vaccine 2007; 25: 5301–5307.

    Article  CAS  PubMed  Google Scholar 

  29. Santosuosso M, McCormick S, Zhang X, Zganiacz A, Xing Z . Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect Immun 2006; 74: 4634–4643.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zeng M, Xu Q, Hesek ED, Pichichero ME . N-fragment of edema factor as a candidate antigen for immunization against anthrax. Vaccine 2006; 24: 662–670.

    Article  CAS  PubMed  Google Scholar 

  31. Zeng M, Xu Q, Elias M, Pichichero ME, Simpson LL, Smith LA . Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine. Vaccine 2007; 25: 7540–7548.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS et al. Botulinum toxin as a biological weapon. In: Henderson DA, Inglesby TV, O' Toole T (eds). Bioterrorism: Guidelines for Medical and Public Health Management. AMA Press: Chicago, IL, 2002, pp 141–165.

    Google Scholar 

  33. Caya JG, Agni R, Miller JE . Clostridium botulinum and the clinical laboratorian: a detailed review of botulism, including biological warfare ramifications of botulinum toxin. Arch Pathol Lab Med 2004; 128: 653–662.

    CAS  PubMed  Google Scholar 

  34. Fujihashi K, Staats HF, Kozaki S, Pascual DW . Mucosal vaccine development for botulinum intoxication. Expert Rev Vaccines 2007; 6: 35–45.

    Article  CAS  PubMed  Google Scholar 

  35. Kobayashi R, Kohda T, Kataoka K, Ihara H, Kozaki S, Pascual DW et al. A novel neurotoxoid vaccine prevents mucosal botulism. J Immunol 2005; 174: 2190–2195.

    Article  CAS  PubMed  Google Scholar 

  36. Maddaloni M, Staats HF, Mierzejewska D, Hoyt T, Robinson A, Callis G et al. Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A. J Immunol 2006; 177: 5524–5532.

    Article  CAS  PubMed  Google Scholar 

  37. Lemiale F, Kong WP, Akyurek LM, Ling X, Huang Y, Chakrabarti BK et al. Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol 2003; 77: 10078–10087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Casimiro DR, Chen L, Fu TM, Evans RK, Caulfield MJ, Davies ME et al. Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 2003; 77: 6305–6313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yang Y, Li Q, Ertl HC, Wilson JM . Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995; 69: 2004–2015.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Barouch DH, McKay PF, Sumida SM, Santra S, Jackson SS, Gorgone DA et al. Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J Virol 2003; 77: 8729–8735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Papp Z, Babiuk LA, Baca-Estrada ME . The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1. Vaccine 1999; 17: 933–943.

    Article  CAS  PubMed  Google Scholar 

  42. Fischer L, Tronel JP, Pardo-David C, Tanner P, Colombet G, Minke J et al. Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge. Vaccine 2002; 20: 3485–3497.

    Article  CAS  PubMed  Google Scholar 

  43. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B . A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998; 95: 2509–2514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zeng M, Smith SK, Siegel F, Shi Z, Van Kampen KR, Elmets CA et al. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination. Biotechniques 2001; 31: 260–262.

    Article  CAS  PubMed  Google Scholar 

  45. Kimura K, Fujii N, Tsuzuki K, Murakami T, Indoh T, Yokosawa N et al. The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome. Biochem Biophys Res Commun 1990; 171: 1304–1311.

    Article  CAS  PubMed  Google Scholar 

  46. Singh SR, Hulett K, Pillai SR, Dennis VA, Oh MK, Scissum-Gunn K . Mucosal immunization with recombinant MOMP genetically linked with modified cholera toxin confers protection against Chlamydia trachomatis infection. Vaccine 2006; 24: 1213–1224.

    Article  CAS  PubMed  Google Scholar 

  47. Russell MW, Wu HY . Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit. Infect Immun 1991; 59: 4061–4070.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zeng M, Xu Q, Pichichero ME . Protection against anthrax by needle-free mucosal immunization with human anthrax vaccine. Vaccine 2007; 25: 3558–3594.

    Google Scholar 

  49. Watanabe I, Hagiwara Y, Kadowaki SE, Yoshikawa T, Komase K, Aizawa C et al. Characterization of protective immune responses induced by nasal influenza vaccine containing mutant cholera toxin as a safe adjuvant (CT112K). Vaccine 2002; 20: 3443–3455.

    Article  CAS  PubMed  Google Scholar 

  50. Arimitsu H, Lee JC, Sakaguchi Y, Hayakawa Y, Hayashi M, Nakaura M et al. Vaccination with recombinant whole heavy chain fragments of Clostridium botulinum Type C and D neurotoxins. Clin Diagn Lab Immunol 2004; 11: 496–502.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Nowakowski A, Wang C, Powers DB, Amersdorfer P, Smith TJ, Montgomery VA et al. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proc Natl Acad Sci USA 2002; 99: 11346–11350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Shi Z, Zeng M, Yang G, Siegel F, Cain LJ, van Kampen KR et al. Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines. J Virol 2001; 75: 11474–11482.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Harvey BG, Hackett NR, El-Sawy T, Rosengart TK, Hirschowitz EA, Lieberman MD et al. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol 1999; 73: 6729–6742.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Zabner J, Ramsey BW, Meeker DP, Aitken ML, Balfour RP, Gibson RL et al. Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin Invest 1996; 97: 1504–1511.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the US Public Service research Grant AI055946 (MZ) from the National Institute of Allergy and Infectious Diseases. ME and LLS were supported by National Institutes of Health Contract NO1-AI30028, Grants NS022153 and GM57345. We are grateful to Eric D Hesek for constructing and purifying the adenoviral vectors.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    Q Xu, M E Pichichero & M Zeng

  2. Department of Medicine and Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

    L L Simpson & Md Elias

  3. Integrated Toxicology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA

    L A Smith

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  1. Q Xu
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  2. M E Pichichero
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Correspondence to M Zeng.

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Xu, Q., Pichichero, M., Simpson, L. et al. An adenoviral vector-based mucosal vaccine is effective in protection against botulism. Gene Ther 16, 367–375 (2009). https://doi.org/10.1038/gt.2008.181

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