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. 1996 Aug;64(8):3168–3173. doi: 10.1128/iai.64.8.3168-3173.1996

Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin.

R Anderson 1, X M Gao 1, A Papakonstantinopoulou 1, M Roberts 1, G Dougan 1
PMCID: PMC174203  PMID: 8757849

Abstract

Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani. Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin. A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae. A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed. Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC. The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model. Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC. The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon. Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin. Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin.

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Selected References

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  1. Angus P. W., Mihaly G. W., Morgan D. J., Smallwood R. A. Oxygen dependence of omeprazole clearance and sulfone and sulfide metabolite formation in the isolated perfused rat liver. J Pharmacol Exp Ther. 1989 Sep;250(3):1043–1047. [PubMed] [Google Scholar]
  2. Barry M. A., Lai W. C., Johnston S. A. Protection against mycoplasma infection using expression-library immunization. Nature. 1995 Oct 19;377(6550):632–635. doi: 10.1038/377632a0. [DOI] [PubMed] [Google Scholar]
  3. Charles I. G., Rodgers B. C., Makoff A. J., Chatfield S. N., Slater D. E., Fairweather N. F. Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system. Infect Immun. 1991 May;59(5):1627–1632. doi: 10.1128/iai.59.5.1627-1632.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chatfield S. N., Charles I. G., Makoff A. J., Oxer M. D., Dougan G., Pickard D., Slater D., Fairweather N. F. Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine. Biotechnology (N Y) 1992 Aug;10(8):888–892. doi: 10.1038/nbt0892-888. [DOI] [PubMed] [Google Scholar]
  5. Chatfield S. N., Fairweather N., Charles I., Pickard D., Levine M., Hone D., Posada M., Strugnell R. A., Dougan G. Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine. Vaccine. 1992;10(1):53–60. doi: 10.1016/0264-410x(92)90420-o. [DOI] [PubMed] [Google Scholar]
  6. Cheng L., Ziegelhoffer P. R., Yang N. S. In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4455–4459. doi: 10.1073/pnas.90.10.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clare J. J., Rayment F. B., Ballantine S. P., Sreekrishna K., Romanos M. A. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology (N Y) 1991 May;9(5):455–460. doi: 10.1038/nbt0591-455. [DOI] [PubMed] [Google Scholar]
  8. Conry R. M., LoBuglio A. F., Kantor J., Schlom J., Loechel F., Moore S. E., Sumerel L. A., Barlow D. L., Abrams S., Curiel D. T. Immune response to a carcinoembryonic antigen polynucleotide vaccine. Cancer Res. 1994 Mar 1;54(5):1164–1168. [PubMed] [Google Scholar]
  9. Conry R. M., LoBuglio A. F., Loechel F., Moore S. E., Sumerel L. A., Barlow D. L., Curiel D. T. A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity. Gene Ther. 1995 Jan;2(1):59–65. [PubMed] [Google Scholar]
  10. Cox G. J., Zamb T. J., Babiuk L. A. Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA. J Virol. 1993 Sep;67(9):5664–5667. doi: 10.1128/jvi.67.9.5664-5667.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Danko I., Fritz J. D., Jiao S., Hogan K., Latendresse J. S., Wolff J. A. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther. 1994 Mar;1(2):114–121. [PubMed] [Google Scholar]
  12. Davis H. L., Michel M. L., Whalen R. G. DNA-based immunization induces continuous secretion of hepatitis B surface antigen and high levels of circulating antibody. Hum Mol Genet. 1993 Nov;2(11):1847–1851. doi: 10.1093/hmg/2.11.1847. [DOI] [PubMed] [Google Scholar]
  13. Davis H. L., Schirmbeck R., Reimann J., Whalen R. G. DNA-mediated immunization in mice induces a potent MHC class I-restricted cytotoxic T lymphocyte response to the hepatitis B envelope protein. Hum Gene Ther. 1995 Nov;6(11):1447–1456. doi: 10.1089/hum.1995.6.11-1447. [DOI] [PubMed] [Google Scholar]
  14. Donnelly J. J., Friedman A., Martinez D., Montgomery D. L., Shiver J. W., Motzel S. L., Ulmer J. B., Liu M. A. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus. Nat Med. 1995 Jun;1(6):583–587. doi: 10.1038/nm0695-583. [DOI] [PubMed] [Google Scholar]
  15. Fairweather N. F., Lyness V. A., Maskell D. J. Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli. Infect Immun. 1987 Nov;55(11):2541–2545. doi: 10.1128/iai.55.11.2541-2545.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Figueiredo D., Turcotte C., Frankel G., Li Y., Dolly O., Wilkin G., Marriott D., Fairweather N., Dougan G. Characterization of recombinant tetanus toxin derivatives suitable for vaccine development. Infect Immun. 1995 Aug;63(8):3218–3221. doi: 10.1128/iai.63.8.3218-3221.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fuller D. H., Haynes J. R. A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 120 vaccine. AIDS Res Hum Retroviruses. 1994 Nov;10(11):1433–1441. doi: 10.1089/aid.1994.10.1433. [DOI] [PubMed] [Google Scholar]
  18. Fynan E. F., Robinson H. L., Webster R. G. Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. DNA Cell Biol. 1993 Nov;12(9):785–789. doi: 10.1089/dna.1993.12.785. [DOI] [PubMed] [Google Scholar]
  19. Fynan E. F., Webster R. G., Fuller D. H., Haynes J. R., Santoro J. C., Robinson H. L. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11478–11482. doi: 10.1073/pnas.90.24.11478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Halpern J. L., Loftus A. Characterization of the receptor-binding domain of tetanus toxin. J Biol Chem. 1993 May 25;268(15):11188–11192. [PubMed] [Google Scholar]
  21. Helting T. B., Zwisler O. Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments. J Biol Chem. 1977 Jan 10;252(1):187–193. [PubMed] [Google Scholar]
  22. Helting T. B., Zwisler O., Wiegandt H. Structure of tetanus toxin. II. Toxin binding to ganglioside. J Biol Chem. 1977 Jan 10;252(1):194–198. [PubMed] [Google Scholar]
  23. Justewicz D. M., Morin M. J., Robinson H. L., Webster R. G. Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin. J Virol. 1995 Dec;69(12):7712–7717. doi: 10.1128/jvi.69.12.7712-7717.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lagging L. M., Meyer K., Hoft D., Houghton M., Belshe R. B., Ray R. Immune responses to plasmid DNA encoding the hepatitis C virus core protein. J Virol. 1995 Sep;69(9):5859–5863. doi: 10.1128/jvi.69.9.5859-5863.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li Y., Foran P., Fairweather N. F., de Paiva A., Weller U., Dougan G., Dolly J. O. A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain. Biochemistry. 1994 Jun 7;33(22):7014–7020. doi: 10.1021/bi00188a034. [DOI] [PubMed] [Google Scholar]
  26. Lowrie D. B., Tascon R. E., Colston M. J., Silva C. L. Towards a DNA vaccine against tuberculosis. Vaccine. 1994 Dec;12(16):1537–1540. doi: 10.1016/0264-410x(94)90080-9. [DOI] [PubMed] [Google Scholar]
  27. Lu S., Santoro J. C., Fuller D. H., Haynes J. R., Robinson H. L. Use of DNAs expressing HIV-1 Env and noninfectious HIV-1 particles to raise antibody responses in mice. Virology. 1995 May 10;209(1):147–154. doi: 10.1006/viro.1995.1238. [DOI] [PubMed] [Google Scholar]
  28. Manickan E., Rouse R. J., Yu Z., Wire W. S., Rouse B. T. Genetic immunization against herpes simplex virus. Protection is mediated by CD4+ T lymphocytes. J Immunol. 1995 Jul 1;155(1):259–265. [PubMed] [Google Scholar]
  29. Mor G., Klinman D. M., Shapiro S., Hagiwara E., Sedegah M., Norman J. A., Hoffman S. L., Steinberg A. D. Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA. J Immunol. 1995 Aug 15;155(4):2039–2046. [PubMed] [Google Scholar]
  30. Morris N. P., Consiglio E., Kohn L. D., Habig W. H., Hardegree M. C., Helting T. B. Interaction of fragments B and C of tetanus toxin with neural and thyroid membranes and with gangliosides. J Biol Chem. 1980 Jul 10;255(13):6071–6076. [PubMed] [Google Scholar]
  31. Raz E., Watanabe A., Baird S. M., Eisenberg R. A., Parr T. B., Lotz M., Kipps T. J., Carson D. A. Systemic immunological effects of cytokine genes injected into skeletal muscle. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4523–4527. doi: 10.1073/pnas.90.10.4523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Romanos M. A., Makoff A. J., Fairweather N. F., Beesley K. M., Slater D. E., Rayment F. B., Payne M. M., Clare J. J. Expression of tetanus toxin fragment C in yeast: gene synthesis is required to eliminate fortuitous polyadenylation sites in AT-rich DNA. Nucleic Acids Res. 1991 Apr 11;19(7):1461–1467. doi: 10.1093/nar/19.7.1461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schiavo G., Ferrari G., Rossetto O., Montecucco C. Tetanus toxin receptor. Specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells. FEBS Lett. 1991 Sep 23;290(1-2):227–230. doi: 10.1016/0014-5793(91)81266-b. [DOI] [PubMed] [Google Scholar]
  34. Sedegah M., Hedstrom R., Hobart P., Hoffman S. L. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9866–9870. doi: 10.1073/pnas.91.21.9866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tang D. C., DeVit M., Johnston S. A. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992 Mar 12;356(6365):152–154. doi: 10.1038/356152a0. [DOI] [PubMed] [Google Scholar]
  36. Tite J. P., Gao X. M., Hughes-Jenkins C. M., Lipscombe M., O'Callaghan D., Dougan G., Liew F. Y. Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier. Immunology. 1990 Aug;70(4):540–546. [PMC free article] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ulmer J. B., Donnelly J. J., Parker S. E., Rhodes G. H., Felgner P. L., Dwarki V. J., Gromkowski S. H., Deck R. R., DeWitt C. M., Friedman A. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science. 1993 Mar 19;259(5102):1745–1749. doi: 10.1126/science.8456302. [DOI] [PubMed] [Google Scholar]
  39. Wang B., Ugen K. E., Srikantan V., Agadjanyan M. G., Dang K., Refaeli Y., Sato A. I., Boyer J., Williams W. V., Weiner D. B. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4156–4160. doi: 10.1073/pnas.90.9.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Webster R. G., Fynan E. F., Santoro J. C., Robinson H. Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin. Vaccine. 1994 Dec;12(16):1495–1498. doi: 10.1016/0264-410x(94)90071-x. [DOI] [PubMed] [Google Scholar]
  41. Wolff J. A., Ludtke J. J., Acsadi G., Williams P., Jani A. Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet. 1992 Sep;1(6):363–369. doi: 10.1093/hmg/1.6.363. [DOI] [PubMed] [Google Scholar]
  42. Wolff J. A., Williams P., Acsadi G., Jiao S., Jani A., Chong W. Conditions affecting direct gene transfer into rodent muscle in vivo. Biotechniques. 1991 Oct;11(4):474–485. [PubMed] [Google Scholar]
  43. Xiang Z. Q., Spitalnik S. L., Cheng J., Erikson J., Wojczyk B., Ertl H. C. Immune responses to nucleic acid vaccines to rabies virus. Virology. 1995 Jun 1;209(2):569–579. doi: 10.1006/viro.1995.1289. [DOI] [PubMed] [Google Scholar]
  44. Xiang Z. Q., Spitalnik S., Tran M., Wunner W. H., Cheng J., Ertl H. C. Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virology. 1994 Feb 15;199(1):132–140. doi: 10.1006/viro.1994.1105. [DOI] [PubMed] [Google Scholar]
  45. Xu D., Liew F. Y. Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major. Immunology. 1995 Feb;84(2):173–176. [PMC free article] [PubMed] [Google Scholar]
  46. Yang W., Waine G. J., McManus D. P. Antibodies to Schistosoma japonicum (Asian bloodfluke) paramyosin induced by nucleic acid vaccination. Biochem Biophys Res Commun. 1995 Jul 26;212(3):1029–1039. doi: 10.1006/bbrc.1995.2073. [DOI] [PubMed] [Google Scholar]
  47. Zarozinski C. C., Fynan E. F., Selin L. K., Robinson H. L., Welsh R. M. Protective CTL-dependent immunity and enhanced immunopathology in mice immunized by particle bombardment with DNA encoding an internal virion protein. J Immunol. 1995 Apr 15;154(8):4010–4017. [PubMed] [Google Scholar]

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