Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Jul;63(7):2689–2696. doi: 10.1128/iai.63.7.2689-2696.1995

Heterologous antigen expression in Vibrio cholerae vector strains.

J R Butterton 1, D T Beattie 1, C L Gardel 1, P A Carroll 1, T Hyman 1, K P Killeen 1, J J Mekalanos 1, S B Calderwood 1
PMCID: PMC173360  PMID: 7790086

Abstract

Live attenuated vector strains of Vibrio cholerae were derived from Peru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin genetic element and attRS1 sequences, which was developed as a live, oral vaccine strain. A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V. cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter. slt-IB was also placed under transcriptional control of the V. cholerae heat shock promoter, htpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respectively. A new technique was used to perform allelic exchange with lacZ. This method uses plasmid p6891MCS, a pBR327 derivative containing cloned V. cholerae lacZ, to insert markers of interest into the V. cholerae chromosome. Recombinants can be detected by simple color screening and antibiotic selection. In vitro measurements of Slt-IB produced by the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene for Slt-IB increased expression over a single copy. The V. cholerae vectors colonized the gastrointestinal mucosa of rabbits after oral immunization, as demonstrated by very high serum antibody responses to V. cholerae antigens. Comparison of the serologic responses to the B subunit of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulated by htpGp, suggested that both the cholera toxin and heat shock promoters were active in vivo, provoking comparable immunologic responses. Orogastric inoculation of rabbits with vector strains evoked serum immunoglobulin G (IgG) responses to Slt-IB in two of the four strains tested; all four strains produced biliary IgA responses. No correlation was observed between the type of promoter expressing slt-IB and the level of serum IgG or biliary IgA response, but the vector strain containing two copies of the gene for slt-IB evoked greater serum IgG responses than strains containing a single copy, consistent with the increased expression of Slt-IB from this strain observed in vitro. A comparison of the serum and biliary antibody responses to Slt-IB expressed from htpGp versus CtxB expressed from the same promoter suggested that CtxB is a more effective orally delivered immunogen.

Full Text

The Full Text of this article is available as a PDF (307.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baudry B., Fasano A., Ketley J., Kaper J. B. Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae. Infect Immun. 1992 Feb;60(2):428–434. doi: 10.1128/iai.60.2.428-434.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blomfield I. C., Vaughn V., Rest R. F., Eisenstein B. I. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol. 1991 Jun;5(6):1447–1457. doi: 10.1111/j.1365-2958.1991.tb00791.x. [DOI] [PubMed] [Google Scholar]
  3. Boyd B., Richardson S., Gariepy J. Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin. Infect Immun. 1991 Mar;59(3):750–757. doi: 10.1128/iai.59.3.750-757.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Butterton J. R., Boyko S. A., Calderwood S. B. Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains. Vaccine. 1993 Oct;11(13):1327–1335. doi: 10.1016/0264-410x(93)90103-5. [DOI] [PubMed] [Google Scholar]
  5. Calderwood S. B., Acheson D. W., Goldberg M. B., Boyko S. A., Donohue-Rolfe A. A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit. Infect Immun. 1990 Sep;58(9):2977–2982. doi: 10.1128/iai.58.9.2977-2982.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Camilli A., Beattie D. T., Mekalanos J. J. Use of genetic recombination as a reporter of gene expression. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2634–2638. doi: 10.1073/pnas.91.7.2634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cray W. C., Jr, Tokunaga E., Pierce N. F. Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae O1. Infect Immun. 1983 Aug;41(2):735–741. doi: 10.1128/iai.41.2.735-741.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Czerkinsky C., Svennerholm A. M., Quiding M., Jonsson R., Holmgren J. Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans. Infect Immun. 1991 Mar;59(3):996–1001. doi: 10.1128/iai.59.3.996-1001.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donnenberg M. S., Kaper J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310–4317. doi: 10.1128/iai.59.12.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Donohue-Rolfe A., Kelley M. A., Bennish M., Keusch G. T. Enzyme-linked immunosorbent assay for shigella toxin. J Clin Microbiol. 1986 Jul;24(1):65–68. doi: 10.1128/jcm.24.1.65-68.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donohue-Rolfe A., Keusch G. T., Edson C., Thorley-Lawson D., Jacewicz M. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J Exp Med. 1984 Dec 1;160(6):1767–1781. doi: 10.1084/jem.160.6.1767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldberg I., Mekalanos J. J. Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. J Bacteriol. 1986 Mar;165(3):715–722. doi: 10.1128/jb.165.3.715-722.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goldberg I., Mekalanos J. J. Effect of a recA mutation on cholera toxin gene amplification and deletion events. J Bacteriol. 1986 Mar;165(3):723–731. doi: 10.1128/jb.165.3.723-731.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldberg M. B., Boyko S. A., Calderwood S. B. Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1125–1129. doi: 10.1073/pnas.88.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goldberg M. B., DiRita V. J., Calderwood S. B. Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis. Infect Immun. 1990 Jan;58(1):55–60. doi: 10.1128/iai.58.1.55-60.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Greatorex J. S., Thorne G. M. Humoral immune responses to Shiga-like toxins and Escherichia coli O157 lipopolysaccharide in hemolytic-uremic syndrome patients and healthy subjects. J Clin Microbiol. 1994 May;32(5):1172–1178. doi: 10.1128/jcm.32.5.1172-1178.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harari I., Arnon R. Carboxy-terminal peptides from the B subunit of Shiga toxin induce a local and parenteral protective effect. Mol Immunol. 1990 Jul;27(7):613–621. doi: 10.1016/0161-5890(90)90003-i. [DOI] [PubMed] [Google Scholar]
  18. Harari I., Donohue-Rolfe A., Keusch G., Arnon R. Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity. Infect Immun. 1988 Jun;56(6):1618–1624. doi: 10.1128/iai.56.6.1618-1624.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herrington D. A., Hall R. H., Losonsky G., Mekalanos J. J., Taylor R. K., Levine M. M. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988 Oct 1;168(4):1487–1492. doi: 10.1084/jem.168.4.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hovde C. J., Calderwood S. B., Mekalanos J. J., Collier R. J. Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2568–2572. doi: 10.1073/pnas.85.8.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Iwanaga M., Yamamoto K., Higa N., Ichinose Y., Nakasone N., Tanabe M. Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol Immunol. 1986;30(11):1075–1083. doi: 10.1111/j.1348-0421.1986.tb03037.x. [DOI] [PubMed] [Google Scholar]
  22. Levine M. M., Black R. E., Clements M. L., Cisneros L., Nalin D. R., Young C. R. Duration of infection-derived immunity to cholera. J Infect Dis. 1981 Jun;143(6):818–820. doi: 10.1093/infdis/143.6.818. [DOI] [PubMed] [Google Scholar]
  23. Levine M. M., Kaper J. B., Herrington D., Ketley J., Losonsky G., Tacket C. O., Tall B., Cryz S. Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet. 1988 Aug 27;2(8609):467–470. doi: 10.1016/s0140-6736(88)90120-1. [DOI] [PubMed] [Google Scholar]
  24. Levine M. M., McEwen J., Losonsky G., Reymann M., Harari I., Brown J. E., Taylor D. N., Donohue-Rolfe A., Cohen D., Bennish M. Antibodies to shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteriae 1 dysentery. J Clin Microbiol. 1992 Jul;30(7):1636–1641. doi: 10.1128/jcm.30.7.1636-1641.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mekalanos J. J. Duplication and amplification of toxin genes in Vibrio cholerae. Cell. 1983 Nov;35(1):253–263. doi: 10.1016/0092-8674(83)90228-3. [DOI] [PubMed] [Google Scholar]
  26. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  27. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Owen R. L., Pierce N. F., Apple R. T., Cray W. C., Jr M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration. J Infect Dis. 1986 Jun;153(6):1108–1118. doi: 10.1093/infdis/153.6.1108. [DOI] [PubMed] [Google Scholar]
  29. Parsot C. Identification of a lacZ gene in Vibrio cholerae. Res Microbiol. 1992 Jan;143(1):27–36. doi: 10.1016/0923-2508(92)90031-i. [DOI] [PubMed] [Google Scholar]
  30. Parsot C., Mekalanos J. J. Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9898–9902. doi: 10.1073/pnas.87.24.9898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pearson G. D., Woods A., Chiang S. L., Mekalanos J. J. CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3750–3754. doi: 10.1073/pnas.90.8.3750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pierce N. F., Cray W. C., Jr, Kaper J. B., Mekalanos J. J. Determinants of immunogenicity and mechanisms of protection by virulent and mutant Vibrio cholerae O1 in rabbits. Infect Immun. 1988 Jan;56(1):142–148. doi: 10.1128/iai.56.1.142-148.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sciortino C. V., Finkelstein R. A. Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo. Infect Immun. 1983 Dec;42(3):990–996. doi: 10.1128/iai.42.3.990-996.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Svennerholm A. M., Sack D. A., Holmgren J., Bardhan P. K. Intestinal antibody responses after immunisation with cholera B subunit. Lancet. 1982 Feb 6;1(8267):305–308. doi: 10.1016/s0140-6736(82)91568-9. [DOI] [PubMed] [Google Scholar]
  35. Swerdlow D. L., Ries A. A. Cholera in the Americas. Guidelines for the clinician. JAMA. 1992 Mar 18;267(11):1495–1499. [PubMed] [Google Scholar]
  36. Swerdlow D. L., Ries A. A. Cholera in the Americas. Guidelines for the clinician. JAMA. 1992 Mar 18;267(11):1495–1499. [PubMed] [Google Scholar]
  37. Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
  38. Taylor D. N., Killeen K. P., Hack D. C., Kenner J. R., Coster T. S., Beattie D. T., Ezzell J., Hyman T., Trofa A., Sjogren M. H. Development of a live, oral, attenuated vaccine against El Tor cholera. J Infect Dis. 1994 Dec;170(6):1518–1523. doi: 10.1093/infdis/170.6.1518. [DOI] [PubMed] [Google Scholar]
  39. Trucksis M., Galen J. E., Michalski J., Fasano A., Kaper J. B. Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5267–5271. doi: 10.1073/pnas.90.11.5267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Waldor M. K., Mekalanos J. J. Emergence of a new cholera pandemic: molecular analysis of virulence determinants in Vibrio cholerae O139 and development of a live vaccine prototype. J Infect Dis. 1994 Aug;170(2):278–283. doi: 10.1093/infdis/170.2.278. [DOI] [PubMed] [Google Scholar]
  41. Weisz-Carrington P., Roux M. E., McWilliams M., PHILLIPS-Quagliata J. M., Lamm M. E. Organ and isotype distribution of plasma cells producing specific antibody after oral immunization: evidence for a generalized secretory immune system. J Immunol. 1979 Oct;123(4):1705–1708. [PubMed] [Google Scholar]
  42. Winner L., 3rd, Mack J., Weltzin R., Mekalanos J. J., Kraehenbuhl J. P., Neutra M. R. New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection. Infect Immun. 1991 Mar;59(3):977–982. doi: 10.1128/iai.59.3.977-982.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES