Skip to main content
PMC home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 1999 Mar 12;48(1):87–100. doi: 10.1016/0378-1135(95)00141-7

Characterization of transmissible gastroenteritis coronavirus S protein expression products in avirulent S. typhimurium Δcya Δcrp: persistence, stability and immune response in swine

Cristian Smerdou a, Alicia Urniza b, Roy Curtis III c, Luis Enjuanes a,∗
PMCID: PMC7117405  PMID: 8701580

Abstract

The spike protein from transmissible gastroenteritis virus (TGEV) was expressed in attenuated S. typhimurium Δcya Δcrp Δasd χ3987. Three partially overlapping fragments of TGEV S gene, encoding the amino-terminal, intermediate, and carboxy-terminal end of the protein, as well as the full length gene were inserted into the asd+ plasmid pYA292 to generate recombinant plasmids pYATS-1, pYATS-2, pYATS-3, and pYATS-4, respectively, which were transformed into S. typhimurium χ3987. Recombinant S. typhimurium χ3987 (pYATS-1) and χ3987 (pYATS-4) expressing constitutively a 53 kDa amino-terminal fragment of the S protein and the full length protein (144 kDa), respectively, showed high stability. After 50 generations in vitro 60% and 20% of the bacteria transformed with pYATS-1 and pYATS-4, respectively, expressed the S-protein antigen. Since S. typhimurium χ3987 (pYATS-1) showed a better level of expression and stability in vitro, this recombinant strain was selected as a potential bivalent vector to induce both immunity to Salmonella and TGEV in swine. In order to study colonization of swine tissues by S. typhimurium Δcya Δcrp, a gene conferring resistance to rifampicin was cloned into the chromosome of S. typhimurium χ3987, generating χ4509 strain. Both S. typhimurium χ4509 (pYA292) and χ4509 (pYATS-1) colonized the ileum of orally inoculated swine with clearance of bacteria between days 10–20 post-infection. The expression of the amino-terminal fragment of the S protein diminished the ability of S. typhimurium χ4509 (pYATS-1) to colonize deep tissues. The recombinant strain S. typhimurium χ3987 (pYATS-1) induced TGEV specific antibodies in both serum and saliva of orally inoculated swine. This strain, as well as S. typhimurium χ3987 (pYA292), also elicited both systemic and mucosal immunity to Salmonella antigens.

Keywords: TGEV, Transmissible gastroenteritis virus; Coronavirus; Swine; Mucosal immunity; Salmonella typhimurium

References

  1. Bacon G.A., Burrows T.W., Yates M. The effects of biochemical mutation on the virulence of Bacterium typhosum: The virulence of mutants. Brit. J. Exp. Pathol. 1950;32:714–724. [PMC free article] [PubMed] [Google Scholar]
  2. Bacon G.A., Burrows T.W., Yates M. The effects of biochemical mutation on the virulence of Bacterium typhosum: The loss of virulence of certain mutants. Brit. J. Exp. Pathol. 1951;32:85–86. [PMC free article] [PubMed] [Google Scholar]
  3. Bennell M.A., Watson D.L. The interactions of porcine and ovine, serum and colostral immunoglobulins with Staphylococcal protein A. Microbiol. Immunol. 1980;24:871–878. doi: 10.1111/j.1348-0421.1980.tb02891.x. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H.C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research. 1979;7:1513–1517. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Correa I., Gebauer F., Bullido M.J., Suñé C., Baay M.F.D., Zwaagstra K.A., Posthumus W.P.A., Lenstra J.A., Enjuanes L. Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus. J. Gen. Virol. 1990;71:271–279. doi: 10.1099/0022-1317-71-2-271. [DOI] [PubMed] [Google Scholar]
  6. Correa I., Jiménez G., Suñé C., Bullido M.J., Enjuanes L. Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus. Virus. Res. 1988;10:77–94. doi: 10.1016/0168-1702(88)90059-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curtiss R., III Chromosomal aberrations associated with mutation to bacteriophage resistance in Escherichia coli. J. Bacteriol. 1965;89:28–40. doi: 10.1128/jb.89.1.28-40.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curtiss R., III . Attenuated Salmonella strains as live vectors for the expression of foreign antigens. In: Woodrowand G.C., Levine M.M., editors. New generation vaccines. Marcel Dekker, Inc; New York: 1990. pp. 161–188. [Google Scholar]
  9. Curtiss R., III, Charamella L.J., Stallions D.R., Mays J.A. Parental functions during conjugation in Escherichia coli K-12. Bacteriol. Rev. 1968;32:320–348. [PMC free article] [PubMed] [Google Scholar]
  10. Curtiss R., III, Galan J.E., Nakayama K., Kelly S.M. Stabilization of recombinant avirulent vaccine strains in vivo. Res. Microbiol. 1990;141:797–805. doi: 10.1016/0923-2508(90)90113-5. [DOI] [PubMed] [Google Scholar]
  11. Curtiss R., III, Kelly S.M. Salmonella typhimurium deletion mutants lacking adenylate cyclase and cylic AMP receptor protein are avirulent and immunogenic. Infect. Immun. 1987;55:3035–3043. doi: 10.1128/iai.55.12.3035-3043.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. de Groot R.J., Luytjes W., Horzineck M.C., van der Zeijst B.A.M., Spaan W.J.M., Lenstra J.A. Evidence for a coiled-coil structure in the spike proteins of coronabiruses. J. Mol. Biol. 1987;196:963–966. doi: 10.1016/0022-2836(87)90422-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Delmas B., Gelfi J., Laude H. Antigenic structure of transmissible gastroenteritis virus. II. Domains in the peplomer glycoprotein. J. Gen. Virol. 1986;67:1405–1418. doi: 10.1099/0022-1317-67-7-1405. [DOI] [PubMed] [Google Scholar]
  14. Delmas B., Rasschaert D., Godet M., Gelfi J., Laude H. Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike protein. J. Gen. Virol. 1990;71:1313–1323. doi: 10.1099/0022-1317-71-6-1313. [DOI] [PubMed] [Google Scholar]
  15. Doggett T.A., Jagusztyn-Krynicka E.K., Curtiss R., III Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium. Infect. Immun. 1993;61:1859–1866. doi: 10.1128/iai.61.5.1859-1866.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Enjuanes L., Van der Zeijst B.A.M. Molecular basis of transmissible gastroenteritis coronavirus (TGEV) epidemiology. In: Siddell S.G., editor. The Coronaviridae. Plenum Press; New York: 1995. pp. 337–376. [Google Scholar]
  17. Galan J.E., Nakayama K., Curtiss R. Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene. 1990;94:29–35. doi: 10.1016/0378-1119(90)90464-3. [DOI] [PubMed] [Google Scholar]
  18. Garwes D.J., Lucas M.H., Higgins D.A., Pike B.V., Cartwright S.F. Antigenicity of structural components from porcine transmissible gastroenteritis virus. Vet. Microbiol. 1978;3:179–190. [Google Scholar]
  19. Gebauer F., Posthumus W.A.P., Correa I., Suñé C., Sánchez C.M., Smerdou C., Lenstra J.A., Meloen R., Enjuanes L. Residues involved in the formation of the antigenic sites of the S protein of transmissible gastroenteritis coronavirus. Virology. 1991;183:225–238. doi: 10.1016/0042-6822(91)90135-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Germanier R., Furer E. Immunity in experimental salmonellosis. II. Basis for the avirulence and protective capacity of galE mutants of Salmonella typhimurium. Infect. Immun. 1971;4:663–673. doi: 10.1128/iai.4.6.663-673.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Godet M., L'Haridon R., Vautherot J.F., Laude H. TGEV coronavirus ORF4 encodes a membrane protein that is incorporated into virions. Virol. 1992;188:666–675. doi: 10.1016/0042-6822(92)90521-P. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoiseth S.K., Stocker B.A.D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981;291:238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  23. Jiménez G., Correa I., Melgosa M.P., Bullido M.J., Enjuanes L. Critical epitopes in transmissible gastroenteritis virus neutralization. J. Virol. 1986;60:131–139. doi: 10.1128/jvi.60.1.131-139.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Montgomery P.C., Cohn J., Lally E.T. The induction and characterization of secretory antibodies. Adv. Exp. Med., Biol. 1974;45:453. doi: 10.1007/978-1-4613-4550-3_54. [DOI] [PubMed] [Google Scholar]
  26. Nakayama K., Kelly S.M., Curtiss R., III Construction of an asd+ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Biotechnology. 1988;6:693–697. [Google Scholar]
  27. Posthumus W.P.A., Lenstra J.A., Schaaper W.M.M., van Nieuwstadt A.P., Enjuanes L., Meloen R.H. Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus. J. Virol. 1990;64:3304–3309. doi: 10.1128/jvi.64.7.3304-3309.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Posthumus W.P.A., Meloen R.H., Enjuanes L., Correa I., van Nieuwestadt A., Koch G. Linear neutralizing epitopes on the peplomer protein of coronaviruses. Adv. Exp. Med. Biol. 1990;276:181–188. doi: 10.1007/978-1-4684-5823-7_25. [DOI] [PubMed] [Google Scholar]
  29. Rasschaert D., Laude L. The predicted primary structure of the peplomer protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J. Gen. Virol. 1987;68:1883–1890. doi: 10.1099/0022-1317-68-7-1883. [DOI] [PubMed] [Google Scholar]
  30. Saif L.J., Wesley R.D. Transmissible Gastroenteritis. In: Leman A.D., editor. Diseases of Swine. Wolfe Publishing Ltd; 1992. pp. 362–386. [Google Scholar]
  31. Sambrook J., Fritsch E.F., Maniatis T. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1989. Molecular cloning: a laboratory manual. [Google Scholar]
  32. Sánchez C.M., Gebauer F., Suñé C., Méndez A., Dopazo J., Enjuanes L. Genetic evolution and tropism of transmissible gastroenteritis coronaviruses. Virology. 1992;190:92–105. doi: 10.1016/0042-6822(92)91195-Z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sánchez C.M., Jiménez G., Laviada M.D., Correa I., Suñé C., Bullido M.J., Gebauer F., Smerdou C., Callebaut P., Escribano J.M., Enjuanes L. Antigenic homology among coronaviruses related to transmissible gastroenteritis virus. Virology. 1990;174:410–417. doi: 10.1016/0042-6822(90)90094-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanz A., García-Barreno B., Nogal M.L., Viñuela E., Enjuanes L. Monoclonal antibodies specific for African swine fever virus proteins. J. Virol. 1985;54:199–206. doi: 10.1128/jvi.54.1.199-206.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schödel F., Peterson D., Hughes J., Milich D. Avirulent Salmonella expressing hybrid hepatitis B virus core/pre-S genes for oral vaccination. Vaccine. 1993;11:143. doi: 10.1016/0264-410x(93)90010-u. [DOI] [PubMed] [Google Scholar]
  36. Siddell S., Wege H., Volker M. The biology of coronaviruses. J. Gen. Virol. 1983;64:761–776. doi: 10.1099/0022-1317-64-4-761. [DOI] [PubMed] [Google Scholar]
  37. Spaan W., Cavanagh D., Horzinek M.C. Coronaviruses: structure and genome expression. J. Gen. Virol. 1988;69:2939–2952. doi: 10.1099/0022-1317-69-12-2939. [DOI] [PubMed] [Google Scholar]
  38. Spaan W., Cavanagh D., Horzinek M.C. Coronaviruses. In: van Regenmorteland M.H.V., Neurath A.R., editors. Immunochemistry of Viruses II. The Basis for Serodiagnosis and Vaccines. Elsevier; 1990. pp. 359–379. [Google Scholar]
  39. Stabel T.J., Mayfield J.E., Tabatabai L.B., Wannemuehler M.J. Oral immunization of mice with attenuated Salmonella typhimurium containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus. Infect. Immun. 1990;58:2048–2055. doi: 10.1128/iai.58.7.2048-2055.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stabel T.J., Mayfield J.E., Tabatabai L.B., Wannemuehler M.J. Swine immunity to an attenuated Salmonella typhimurium mutant containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus. Infect. Inmmun. 1991;59:2941–2947. doi: 10.1128/iai.59.9.2941-2947.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stone S.S., Kemeny L.J., Woods R.D., Jensen M.T. Efficacy of isolated colostral IgA, IgG, and IgM(A) to protect neonatal pigs against the coronavirus transmissible gastroenteritis. Am. J. Vet. Res. 1977;38:1285–1288. [PubMed] [Google Scholar]
  42. Sturman L.S., Holmes K.V. The molecular biology of coronaviruses. Adv. Viral Res. 1983;28:35–112. doi: 10.1016/S0065-3527(08)60721-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wesley R.D., Woods R.D., Correa I., Enjuanes L. Lack of protection in vivo with neutralizing monoclonal antibodies to transmissible gastroenteritis virus. Vet. Microbiol. 1988;18:197. doi: 10.1016/0378-1135(88)90087-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yanisch-Perron P., Vieria J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33:103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  45. Zinder N. Lysogenization and superinfection immunity in Salmonella. Virology. 1958;5:291–326. doi: 10.1016/0042-6822(58)90025-4. [DOI] [PubMed] [Google Scholar]

Articles from Veterinary Microbiology are provided here courtesy of Elsevier

RESOURCES