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. 1998 Oct 6;46(1):111–124. doi: 10.1016/S0168-1702(96)01390-1

Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies

Inés M Antón a,1, Silvia González a, María J Bullido a, Marta Corsín a, Cristina Risco a, Jan PM Langeveld b, Luis Enjuanes a,∗
PMCID: PMC7134021  PMID: 9029784

Abstract

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S+N, S+M, and to a minor extent with M+N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Keywords: Coronavirus, TGEV, Antibody synthesis

References

  1. Antón I.M., Suñé C., Meloen R.H., Borrás-Cuesta F., Enjuanes L. A transmissible gastroenteritiss coronavirus nucleoprotein epitope elicits T helper cells that collaborate in the in vitro antibody synthesis to the three major structural viral proteins. Virology. 1995;212:746–751. doi: 10.1006/viro.1995.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellone M., Karachunski P.I., Ostlie N., Lie S., Conti-Tronconi B.M. Preferential pairing of T and B-cells for production of antibodies without covalent association of T and B epitopes. Eur. J. Immunol. 1994;24:799–804. doi: 10.1002/eji.1830240404. [DOI] [PubMed] [Google Scholar]
  3. Bergmann G., McMillan M., Stohlman S. Characterization of the Ld-restricted cytotoxic T-lymphocyte epitope in the mouse hepatitis virus nucleocapsid protein. J. Virol. 1993;67:7041–7049. doi: 10.1128/jvi.67.12.7041-7049.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berthon P., Bernard S., Salmon H., Binns R.M. Kinetics of the in vitro antibody response to transmissible gastroenteritis (TGE) virus from pig mesenteric lymph node cells, using the ELISASPOT and ELISA tests. J. Immunol. Methods. 1990;131:173–182. doi: 10.1016/0022-1759(90)90188-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bohl E.H., Saif L.J. Passive immunity in transmissible gastroenteritis of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes. Infect. Immunol. 1975;11:23–32. doi: 10.1128/iai.11.1.23-32.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boots A.M., Van-Lierop M.J., Kusters J.G., Van-Kooten G.J., Van-der-Zeijst B.A., Hensen E.J. MHC ClassII-restricted T-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV. Immunology. 1991;72:10–14. [PMC free article] [PubMed] [Google Scholar]
  7. Boots A.M.H., Benaissatrouw B.J., Hesselink W., Rijke E., Schrier C., Hensen E.J. Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein. Vaccine. 1992;10:119–124. doi: 10.1016/0264-410X(92)90028-I. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brim T.A., VanCott J.L., Lunney J.K., Saif L.J. Lymphocyte proliferation responses of pigs inoculated with transmissible gastroenteritis virus or porcine respiratory coronavirus. Am. J. Vet. Res. 1994;55:494–501. [PubMed] [Google Scholar]
  9. Bullido M.J., Correa I., Jiménez G., Suñé C., Gebauer F., Enjuanes L. Induction of transmissible gastroenteritis coronavirus-neutralizing antibodies in vitro by virus-specific T helper cell hybridomas. J. Gen. Virol. 1989;70:659–672. doi: 10.1099/0022-1317-70-3-659. [DOI] [PubMed] [Google Scholar]
  10. Cavanagh D. Coronavirus IBV: Structural characterization of the spike protein. J. Gen. Virol. 1983;64:2577–2583. doi: 10.1099/0022-1317-64-12-2577. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. De Diego M., Laviada M.D., Enjuanes L., Escribano J.M. Epitope specificity of protective lactogenic immunity against swine transmissible gastroenteritis virus. J. Virol. 1992;66:6502–6508. doi: 10.1128/jvi.66.11.6502-6508.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Eleouet J.F., Rasschaert D., Lambert P., Levy L., Vende P., Laude H. Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology. 1995;206:817–822. doi: 10.1006/viro.1995.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Enjuanes L. Development of protection against coronavirus induced diseases. A review. Adv. Exp. Med. Biol. 1995;380:197–211. doi: 10.1007/978-1-4615-1899-0_34. [DOI] [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. Flory E., Pfleiderer M., Stuhler A., Wege H. Induction of protective immunity against coronavirus-induced encephalomyelitis: evidence for an important role of CD8+ T-cells in vivo. Eur. J. Immunol. 1993;23:1757–1761. doi: 10.1002/eji.1830230804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Geysen N.M., Meloen R.H., Barteling S.J. Vol. 81. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid; pp. 3998–4002. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Godet M., ĹHaridon R., Vautherot J.F., Laude H. TGEV coronavirus ORF4 encodes a membrane protein that is incorporated into virions. Virology. 1992;188:666–675. doi: 10.1016/0042-6822(92)90521-P. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heemskerk M.H.M., Schoemaker H.M., Spaan W.J.M., Boog C.J.P. Predominance of MHC class II-restricted CD4+ cytotoxic T-cells against mouse hepatitis virus A59. Immunology. 1994;84:521–527. [PMC free article] [PubMed] [Google Scholar]
  22. Heinz F.X., Allison S.L., Stiasny K., Schalich J., Holzmann H., Mandl C.W., Kunz C. Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis. Vaccine. 1995;13:1636–1642. doi: 10.1016/0264-410x(95)00133-l. [DOI] [PubMed] [Google Scholar]
  23. Helenius A., Bonsdorff C.-H.V. Semliki Forest virus membrane proteins: preparation and characterization of spike complexes soluble in detergent-free medium. Biochim. Biophys. Acta. 1976;436:895–899. doi: 10.1016/0005-2736(76)90421-1. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Körner H., Schiliephake A., Winter J., Zimprich F., Lassmann H., Sedgwick J., Siddell S.G., Wege H. Nucleocapsid or spike protein-specific CD4+ T-lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T-cells. J. Neuroimmunol. 1991;147:2317–2323. [PubMed] [Google Scholar]
  26. La Bonnardière C., Laude H. High interferon titer in newborn pig intestine during experimentally induced viral enteritis. Infect. Immunol. 1981;32:28–31. doi: 10.1128/iai.32.1.28-31.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Laude H., Chapsal J.M., Gelfi J., Labiau S., Grosclaude J. Antigenic structure of transmissible gastroenteritis virus. I Properties of monoclonal antibodies directed against virion proteins. J. Gen. Virol. 1986;67:119–130. doi: 10.1099/0022-1317-67-1-119. [DOI] [PubMed] [Google Scholar]
  29. Laude H., Charley B., Gelfi J. Replication of transmissible gastroenteritis coronavirus (TGEV) in swine alveolar macrophages. J. Gen. Virol. 1984;65:327–335. doi: 10.1099/0022-1317-65-2-327. [DOI] [PubMed] [Google Scholar]
  30. Lenstra J.A., Kusters J.G., Koch G., van der Zeijst B.A.M. Antigenicity of the peplomer protein of infectious bronchitis virus. Mol. Immunol. 1989;26:7–15. doi: 10.1016/0161-5890(89)90014-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lunney J.K., Pescovitz M.D., Sachs D.H. The swine major histocompatibility complex: its structure and function. In: Tumbleson M.E., editor. Swine in Biomedical Research. Plenum Press; New York: 1986. pp. 1821–1836. [Google Scholar]
  32. McClurkin A.W., Norman J.O. Studies on transmissible gastroenteritis of swine II. Selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis. Can. J. Comp. Vet. Sci. 1966;30:190–198. [PMC free article] [PubMed] [Google Scholar]
  33. Mendez A., Smerdou C., Izeta A., Gebauer F., Enjuanes L. Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity. Virology. 1996;217:495–507. doi: 10.1006/viro.1996.0144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mobley J., Evans G., Dailey M.O., Perlman S. Immune response to a murine coronavirus: identification of a homing receptor-negative CD4+ T-cell subset that responds to viral glycoproteins. Virology. 1992;187:443–452. doi: 10.1016/0042-6822(92)90446-V. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Noelle R.J., Snow E.C. T helper cell-dependent B-cell activation. FASEB J. 1991;5:2770–2776. doi: 10.1096/fasebj.5.13.1833257. [DOI] [PubMed] [Google Scholar]
  36. Pescovitz M.D., Lunney J.L., Sachs D. Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J. Immunol. 1984;133:368–375. [PubMed] [Google Scholar]
  37. Risco C., Antón I.M., Enjuanes L., Carrascosa J.L. The transmissible gastroenteritis coronavirus contains a spherical core shell consisting of M and N-proteins. J. Virol. 1996;70:4773–4777. doi: 10.1128/jvi.70.7.4773-4777.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roehrig J.T., Johnson A.J., Hunt A.R., Beaty B.J., Mathews J.H. Enhancement of the antibody response to flavivirus B-cell epitope by using homologous or heterologous T-cell epitopes. J. Virol. 1992;66:3385–3390. doi: 10.1128/jvi.66.6.3385-3390.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rothschild M.F., Hill H.T., Christian L.L., Lie W.R., Warner C.M. Genetic differences in serum neutralization titers of pigs after vaccination with Pseudorabies modified live-virus vaccine. Am. J. Vet. Res. 1984;45:1216–1218. [PubMed] [Google Scholar]
  40. Saalmüller A., Reddehase M.J., Bühring H., Jonjic S., Koszinowski U.H. Simulataneous expression of CD4 and CD8 antigens by a substantial proportion of resting porcine T-lymphocytes. Eur. J. Immunol. 1987;17:1297–1301. doi: 10.1002/eji.1830170912. [DOI] [PubMed] [Google Scholar]
  41. Sachs D., Leight G., Cone J., Schwarz S., Stuart L., Rosemberg S. Transplantation in miniature swine. I. Fixation of the major histocompatibility complex. Transplantation. 1976;22:559–567. doi: 10.1097/00007890-197612000-00004. [DOI] [PubMed] [Google Scholar]
  42. Saif L.J., Bohl E.H. Transmissible gastroenteritis. In: Leman B.S.A.D., Glock R.D., Mengeling W.L., Penny R.H.C., Scholl E., editors. Diseases of Swine. Iowa State University; Ames, Iowa: 1986. pp. 255–274. [Google Scholar]
  43. 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]
  44. Sarobe P., Lasarte J.J., Golvano J.J., Gullón A., Civeira M.P., Prieto J., Borrás-Cuesta F. Induction of antibodies against a peptide hapten does not require covalent linkage between the hapten and a class II presentable peptide. Eur. J. Immunol. 1991;21:1555–1558. doi: 10.1002/eji.1830210633. [DOI] [PubMed] [Google Scholar]
  45. Simons K., Helenius A., Garoff H. Solubilization of the membrane proteins from Semliki Forest virus with Triton X-100. J. Mol. Biol. 1973;80:119–133. doi: 10.1016/0022-2836(73)90236-2. [DOI] [PubMed] [Google Scholar]
  46. Smerdou C., Antón I.M., Plana J., Curtiss R., Enjuanes L. A continuos epitope from Transmissible gastroenteritis coronavirus S-protein fused to E. coli heatlabile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity. Virus Res. 1996;41:1–9. doi: 10.1016/0168-1702(95)01265-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stohlman S.A., Bergmann C., Van der Veen R.C., Hinton D.R. Mouse hepatitis virus-specific cytotoxic T-lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J. Virol. 1995;69:684–694. doi: 10.1128/jvi.69.2.684-694.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stohlman S.A., Kyuwa S., Cohen M., Bergmann C., Polo J.M., Yeh J., Anthony R., Keck J.G. Mouse hepatitis virus nucleocapsid protein-specific cytotoxic T-lymphocytes are Ld restricted and specific for the carboxy terminus. Virology. 1992;189:217–224. doi: 10.1016/0042-6822(92)90697-N. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stone S.S., Stone J., Stone K.L., 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]
  50. Sturman L.S., Holmes K.V., Behnke J. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid. J. Virol. 1980;33:449–462. doi: 10.1128/jvi.33.1.449-462.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Towbin H., Staehlin T., Gordon J. Vol. 76. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications; pp. 4350–4354. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Underdahl N.R., Mebus C.A., Stair E.L., Rhodes M.B., McGill L.D., Twiehaus M.J. Isolation of transmissible gastroenteritis virus from lungs of market-weight swine. Am. J. Vet. Res. 1974;35:1209–1216. [PubMed] [Google Scholar]
  53. Valero R.M., Bray A.M., Campbell R.A., Dipasquale A., Margellis C., Rodda S.J., Geysen H.M., Maeji N.J. Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports. Int. J. Protein. 1993;42:1–9. doi: 10.1111/j.1399-3011.1993.tb00341.x. [DOI] [PubMed] [Google Scholar]
  54. VanCott J.L., Brim T.A., Simkins R.A., Saif L.J. Isotype-specific antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut- and bronchus-associated lymphoid tissues of suckling pigs. J. Immunol. 1993;150:3990–4000. [PubMed] [Google Scholar]
  55. Wege H., Schliephake A., Korner H., Flory E., Wege H. An immunodominant CD4+ T-cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis. J. Gen. Virol. 1993;74:1287–1294. doi: 10.1099/0022-1317-74-7-1287. [DOI] [PubMed] [Google Scholar]
  56. 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;18:197–208. doi: 10.1016/0378-1135(88)90087-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wesley R.D., Woods R.D., Cheung A.K. Genetic basis for the pathogenesis of transmissible gastroenteritis virus. J. Virol. 1990;64:4761–4766. doi: 10.1128/jvi.64.10.4761-4766.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wesseling J.G., Godeke G.J., Schijns V.E.C.J., Prevec L., Frank F.L., Horzinek M.C., Rottier P.J.M. Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirus vector protect mice against a lethal infection. J. Gen. Virol. 1993;74:2061–2069. doi: 10.1099/0022-1317-74-10-2061. [DOI] [PubMed] [Google Scholar]
  59. Xue S., Jaszewski A., Perlman S. Identification of a CD4+ T-cell epitope within the M-protein of a neurotropic coronavirus. Virology. 1995;208:173–179. doi: 10.1006/viro.1995.1140. [DOI] [PMC free article] [PubMed] [Google Scholar]

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