Skip to main content
NCBI home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2005 Dec 6;27(12):3447–3455. doi: 10.1002/eji.1830271245

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice

Alain Lamarre 1,2, Mathilde W N Yu 1, Fanny Chagnon 1, Pierre J Talbot 1,✉
PMCID: PMC7163705  PMID: 9464834

Abstract

The variable region genes of a murine anti‐coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32‐kDa protein was identified by Western immunoblotting with specific rabbit anti‐idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N‐terminal but not with a C‐terminal histidine tag. Purified scFv fragments retained the antigen‐binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen‐binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv‐treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus‐induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half‐life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.

Keywords: Infectious immunity‐virus, Immunotherapy, In vivo animal model, Antibody, Rodent

References

  • 1. Wege, H. , Siddell, S. ,. and Ter Meulen, V. , Curr. Top. Microbiol. Immunol. 1982. 99: 165. [DOI] [PubMed] [Google Scholar]
  • 2. Spaan, W. , Cavanagh, D. and Horzinek, M. C. , in Van Regenmortel M. H. V. and Neurath A. R. (Eds.), Immunochemistry of viruses The basis for serodiagnosis and vaccines, Vol 2, Elsevier Science Publishers B. V, Amsterdam: 1990, p. 359. [Google Scholar]
  • 3. Siddell, S. , Wege, H. and Ter Meulen, V. , J. Gen. Virol. 1983. 64: 761. [DOI] [PubMed] [Google Scholar]
  • 4. Buchmeier, M. J. , Lewicki, H. A. , Talbot, P. J. and Knobler, R. L. , Virology 1984. 132: 261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Daniel, C. and Talbot, P. J. , Virology 1990. 174: 87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Fleming, J. O. , Shubin, R. A. , Sussman, M. A. , Casteel, N. and Stohlman, S. A. , Virology 1989. 168: 162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Lecomte, J. , Cainelli‐Gebara, V. , Mercier, G. , Mansour, S. , Talbot, P. J. , Lussier, G. and Oth, D. , Arch. Virol. 1987. 97: 123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Yokomori, K. , Baker, S. C. , Stohlman, S. A. and Lai, M. M. C. , J. Virol. 1992. 66: 2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Lamarre, A. and Talbot, P. J. , J. Immunol. 1995. 154: 3975. [PubMed] [Google Scholar]
  • 10. Mountain, A. and Adair, J. R. , Biotechnol. Genet. Eng. Rev. 1992. 10: 1. [DOI] [PubMed] [Google Scholar]
  • 11. Lefranc, G. and Lefranc, M. P. , Biochimie 1990. 72: 639. [DOI] [PubMed] [Google Scholar]
  • 12. Ward, E. S. , FASEB J. 1992. 6: 2422. [DOI] [PubMed] [Google Scholar]
  • 13. Chanock, R. M. , Crowe, J. E. Jr. , Murphy, B. R. and Burton, D. R. , Infect. Agents Dis. 1993. 2: 118. [PubMed] [Google Scholar]
  • 14. Daniel, C. and Talbot, P. J. , Arch. Virol. 1987. 96: 241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Mounir, S. and Talbot, P. J. , J. Gen. Virol. 1992. 73: 2731. [DOI] [PubMed] [Google Scholar]
  • 16. Ward, E. S. , Güssow, D. , Griffiths, A. D. , Jones, P. T. and Winter, G. , Nature 1989. 341: 544. [DOI] [PubMed] [Google Scholar]
  • 17. Orlandi, R. , Güssow, D. H. , Jones, P. T. and Winter, G. , Proc. Natl. Acad. Sci. USA 1989. 86: 3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Clackson, T. , Hoogenboom, H. R. , Griffiths, A. D. and Winter, G. , Nature 1991. 352: 624. [DOI] [PubMed] [Google Scholar]
  • 19. McCafferty, J. , Griffiths, A. D. , Winter, G. and Chiswell, D. J. , Nature 1990. 348: 552. [DOI] [PubMed] [Google Scholar]
  • 20. Sanger, F. , Nicklen, S. and Coulson, A. R. , Proc. Natl. Acad. Sci. USA 1977. 74: 5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Laemmli, U. K. , Nature 1970. 227: 680. [DOI] [PubMed] [Google Scholar]
  • 22. Lamarre, A. , Lecomte, J. and Talbot, P. J. , J. Immunol. 1991. 147: 4256. [PubMed] [Google Scholar]
  • 23. Talbot, P. J. , Salmi, A. A. , Knobler, R. L. and Buchmeier, M. J. , Virology 1984. 132: 250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Horton, R. M. , Hunt, H. D. , Ho, S. N. , Pullen, J. K. and Pease, L. R. , Gene 1977. 77: 61. [DOI] [PubMed] [Google Scholar]
  • 25. Huston, J. S. , Levinson, D. , Mudgett‐Hunter, M. , Tai, M. S. , Novotny, J. , Margolies, M. N. , Ridge, R. J. , Bruccoleri, R. E. , Haber, E. , Crea, R. and Oppermann, H. , Proc. Natl. Acad. Sci. USA 1988. 85: 5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Winter, E. , Radbruch, A. and Krawinkel, U. , EMBO J. 1985. 4: 2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Kabat, E. A. , Wu, T. T. , Perry, H. M. , Gottesman, K. S. and Foeller, C. , Sequences of proteins of immunological interest, 5th Ed., National Institutes of Health, Bethesda: 1991, p. 2597. [Google Scholar]
  • 28. Lawler, A. M. , Kearney, J. F. , Kuehl, M. and Gearhart, P. J. , Proc. Natl. Acad. Sci. USA 1989. 86: 6744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Avner, B. , Swindell, L. , Sharp, E. , Liao, S. K. , Ogden, J. R. , Avner, B. P. and Oldham, R. K. , Mol. Biother. 1991. 3: 14. [PubMed] [Google Scholar]
  • 30. Barbas, C. F. III. , Björling, E. , Chiodi, F. , Dunlop, N. , Cababa, D. , Jones, T. M. , Zebedee, S. L. , Persson, M. A. A. , Nara, P. L. , Norrby, E. and Burton, D. R. , Proc. Natl. Acad. Sci. USA 1992. 89: 9339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Cheung, S. C. , Dietzschold, B. , Koprowski, H. , Hotkins, A. L. and Rando, R. F. , J. Virol. 1992. 66: 6714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Barbas, C. F. III. , Crowe, J. E. Jr. , Cababa, D. , Jones, T. M. , Zebedee, S. L. , Murphy, B. R. , Chanock, R. M. and Burton, D. R. , Proc. Natl. Acad. Sci. USA 1992. 89: 10164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Jiang, W. , Bonnert, T. P. , Venugopal, K. and Gould, E. A. , Virology 1994. 200: 21. [DOI] [PubMed] [Google Scholar]
  • 34. Lake, D. F. , Lam, K. S. , Peng, L. and Hersh, E. M. , Mol. Immunol. 1994. 31: 845. [DOI] [PubMed] [Google Scholar]
  • 35. Colcher, D. , Bird, R. , Roselli, M. , Hardman, K. D. , Johnson, S. , Pope, S. , Dodd, S. W. , Pantoliano, M. W. , Milenic, D. E. and Schlom, J. , J. Natl. Cancer Inst. 1990. 82: 1191. [DOI] [PubMed] [Google Scholar]
  • 36. Friedman, P. N. , Chace, D. F. , Trail, P. A. and Siegall, C. B. , J. Immunol. 1993. 150: 3054. [PubMed] [Google Scholar]
  • 37. Laroche, Y. , Demaeyer, M. , Stassen, J. M. , Gansemans, Y. , Demarsin, E. , Matthyssens, G. , Collen, D. and Holvoet, P. , J. Biol. Chem. 1991. 266: 16343. [PubMed] [Google Scholar]
  • 38. Reiter, Y. , Pai, L. H. , Brinkmann, U. , Wang, Q. C. and Pastan, I. , Cancer Res. 1994. 54: 2714. [PubMed] [Google Scholar]
  • 39. Kalinke, U. , Krebber, A. , Krebber, C. , Bucher, E. , Plückthun, A. , Zinkernagel, R. M. and Hengartner, H. , Eur. J. Immunol. 1996. 26: 2801. [DOI] [PubMed] [Google Scholar]
  • 40. Cumber, A. J. , Ward, E. S. , Winter, G. , Parnell, G. D. and Wawrzynczak, E. J. , J. Immunol. 1992. 149: 120. [PubMed] [Google Scholar]
  • 41. Benhar, I. and Pastan, I. , J. Biol. Chem. 1995. 270: 23373. [DOI] [PubMed] [Google Scholar]

Articles from European Journal of Immunology are provided here courtesy of Wiley

RESOURCES