Skip to main content
Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 1991 Jun;84(3):508–514.

Characterization of human monoclonal antibodies directed against the pp65-kD matrix antigen of human cytomegalovirus.

M Ohlin 1, V A Sundqvist 1, G Gilljam 1, U Rudén 1, F O Gombert 1, B Wahren 1, C A Borrebaeck 1
PMCID: PMC1535436  PMID: 1710548

Abstract

Human monoclonal antibodies specific for human cytomegalovirus (CMV) antigens have been established using peripheral blood lymphocytes from a seropositive donor. Immortalization of antigen-specific B cells was achieved by Epstein-Barr virus transformation followed by somatic cell fusion of antigen-specific lymphoblastoid cells. Four clones producing high-affinity antibodies (0.2-7 x 10(9) M-1) specific for the viral matrix protein pp65 have been further characterized with respect to epitope specificity of secreted antibodies. The studied antigen represents a major protein produced by in vitro-cultivated virus, and is important in the serodiagnosis of CMV infection. The human monoclonal antibodies recognized different epitopes, some of which proved to be overlapping. The fine specificity of these antibodies was evaluated using synthetic peptides covering the sequence of pp65. The antibody MO58 recognized a linear epitope (residues 283-288) whereas antibody MO53 recognized a discontinuous epitope involving residues 208-216 and 280-285. Despite the close proximity of these epitopes, the antibodies did not compete with each other for the same binding site on intact antigen.

Full text

PDF
508

Images in this article

Selected References

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

  1. Beatty J. D., Beatty B. G., Vlahos W. G. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods. 1987 Jun 26;100(1-2):173–179. doi: 10.1016/0022-1759(87)90187-6. [DOI] [PubMed] [Google Scholar]
  2. Borrebaeck C. A., Danielsson L., Möller S. A. Human monoclonal antibodies produced by primary in vitro immunization of peripheral blood lymphocytes. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3995–3999. doi: 10.1073/pnas.85.11.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borrebaeck C. A. Strategy for the production of human monoclonal antibodies using in vitro activated B cells. J Immunol Methods. 1989 Oct 24;123(2):157–165. doi: 10.1016/0022-1759(89)90219-6. [DOI] [PubMed] [Google Scholar]
  4. Britt W. J., Auger D. Identification of a 65 000 dalton virion envelope protein of human cytomegalovirus. Virus Res. 1985 Dec;4(1):31–36. doi: 10.1016/0168-1702(85)90018-8. [DOI] [PubMed] [Google Scholar]
  5. Britt W. J., Vugler L. Structural and immunological characterization of the intracellular forms of an abundant 68,000 Mr human cytomegalovirus protein. J Gen Virol. 1987 Jul;68(Pt 7):1897–1907. doi: 10.1099/0022-1317-68-7-1897. [DOI] [PubMed] [Google Scholar]
  6. Carroll W. L., Thielemans K., Dilley J., Levy R. Mouse x human heterohybridomas as fusion partners with human B cell tumors. J Immunol Methods. 1986 May 1;89(1):61–72. doi: 10.1016/0022-1759(86)90032-3. [DOI] [PubMed] [Google Scholar]
  7. Clark B. R., Zaia J. A., Balce-Directo L., Ting Y. P. Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus. J Virol. 1984 Jan;49(1):279–282. doi: 10.1128/jvi.49.1.279-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Foung S. K., Perkins S., Bradshaw P., Rowe J., Rabin L. B., Reyes G. R., Lennette E. T. Human monoclonal antibodies to human cytomegalovirus. J Infect Dis. 1989 Mar;159(3):436–443. doi: 10.1093/infdis/159.3.436. [DOI] [PubMed] [Google Scholar]
  9. Geysen H. M., Meloen R. H., Barteling S. J. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3998–4002. doi: 10.1073/pnas.81.13.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilljam G., Wahren B. Properties of IgG subclasses to human cytomegalovirus. J Virol Methods. 1989 Aug;25(2):139–151. doi: 10.1016/0166-0934(89)90028-1. [DOI] [PubMed] [Google Scholar]
  11. Houghten R. A. General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5131–5135. doi: 10.1073/pnas.82.15.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jahn G., Scholl B. C., Traupe B., Fleckenstein B. The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties. J Gen Virol. 1987 May;68(Pt 5):1327–1337. doi: 10.1099/0022-1317-68-5-1327. [DOI] [PubMed] [Google Scholar]
  13. Jameson B. A., Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci. 1988 Mar;4(1):181–186. doi: 10.1093/bioinformatics/4.1.181. [DOI] [PubMed] [Google Scholar]
  14. Kim K. S., Sapienza V. J., Chen C. M., Wisniewski K. Production and characterization of monoclonal antibodies specific for a glycosylated polypeptide of human cytomegalovirus. J Clin Microbiol. 1983 Aug;18(2):331–343. doi: 10.1128/jcm.18.2.331-343.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Ljungman P., Wahren B., Sundqvist V. A. Lymphocyte proliferation and IgG production with herpesvirus antigens in solid phase. J Virol Methods. 1985 Dec;12(3-4):199–208. doi: 10.1016/0166-0934(85)90130-2. [DOI] [PubMed] [Google Scholar]
  17. Masuho Y., Matsumoto Y., Sugano T., Fujinaga S., Minamishima Y. Human monoclonal antibodies neutralizing human cytomegalovirus. J Gen Virol. 1987 May;68(Pt 5):1457–1461. doi: 10.1099/0022-1317-68-5-1457. [DOI] [PubMed] [Google Scholar]
  18. Ohlin M., Broliden P. A., Danielsson L., Wahren B., Rosen J., Jondal M., Borrebaeck C. A. Human monoclonal antibodies against a recombinant HIV envelope antigen produced by primary in vitro immunization. Characterization and epitope mapping. Immunology. 1989 Nov;68(3):325–331. [PMC free article] [PubMed] [Google Scholar]
  19. Ohlin M., Danielsson L., Carlsson R., Borrebaeck C. A. The effect of leucyl-leucine methyl ester on proliferation and Ig secretion of EBV-transformed human B lymphocytes. Immunology. 1989 Apr;66(4):485–490. [PMC free article] [PubMed] [Google Scholar]
  20. Pereira L., Hoffman M., Cremer N. Electrophoretic analysis of polypeptides immune precipitated from cytomegalovirus-infected cell extracts by human sera. Infect Immun. 1982 Jun;36(3):933–942. doi: 10.1128/iai.36.3.933-942.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pereira L., Hoffman M., Gallo D., Cremer N. Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants. Infect Immun. 1982 Jun;36(3):924–932. doi: 10.1128/iai.36.3.924-932.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pereira L., Hoffman M., Tatsuno M., Dondero D. Polymorphism of human cytomegalovirus glycoproteins characterized by monoclonal antibodies. Virology. 1984 Nov;139(1):73–86. doi: 10.1016/0042-6822(84)90331-3. [DOI] [PubMed] [Google Scholar]
  23. Plotkin S. A., Starr S. E., Friedman H. M., Gönczöl E., Weibel R. E. Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge. J Infect Dis. 1989 May;159(5):860–865. doi: 10.1093/infdis/159.5.860. [DOI] [PubMed] [Google Scholar]
  24. Rasmussen L. E., Nelson R. M., Kelsall D. C., Merigan T. C. Murine monoclonal antibody to a single protein neutralizes the infectivity of human cytomegalovirus. Proc Natl Acad Sci U S A. 1984 Feb;81(3):876–880. doi: 10.1073/pnas.81.3.876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Redmond M. J., Leyritz-Wills M., Winger L., Scraba D. G. The selection and characterization of human monoclonal antibodies to human cytomegalovirus. J Virol Methods. 1986 Aug;14(1):9–24. doi: 10.1016/0166-0934(86)90003-0. [DOI] [PubMed] [Google Scholar]
  26. Rüger B., Klages S., Walla B., Albrecht J., Fleckenstein B., Tomlinson P., Barrell B. Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus. J Virol. 1987 Feb;61(2):446–453. doi: 10.1128/jvi.61.2.446-453.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shimokawa K., Bin X., Furukawa T. Comparative study with monospecific and monoclonal antibodies against a 65 K human cytomegalovirus protein. Arch Virol. 1988;101(1-2):79–86. doi: 10.1007/BF01314653. [DOI] [PubMed] [Google Scholar]
  28. Somogyi T., Michelson S., Masse M. J. Genomic location of a human cytomegalovirus protein with protein kinase activity (PK68). Virology. 1990 Jan;174(1):276–285. doi: 10.1016/0042-6822(90)90075-3. [DOI] [PubMed] [Google Scholar]
  29. Stinski M. F. Synthesis of proteins and glycoproteins in cells infected with human cytomegalovirus. J Virol. 1977 Sep;23(3):751–767. doi: 10.1128/jvi.23.3.751-767.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sundqvist V. A., Wahren B. An interchangeable ELISA for cytomegalovirus antigen and antibody. J Virol Methods. 1981 Apr;2(5):301–312. doi: 10.1016/0166-0934(81)90029-x. [DOI] [PubMed] [Google Scholar]
  31. Thiele D. L., Lipsky P. E. Regulation of cellular function by products of lysosomal enzyme activity: elimination of human natural killer cells by a dipeptide methyl ester generated from L-leucine methyl ester by monocytes or polymorphonuclear leukocytes. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2468–2472. doi: 10.1073/pnas.82.8.2468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Utz U., Britt W., Vugler L., Mach M. Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus. J Virol. 1989 May;63(5):1995–2001. doi: 10.1128/jvi.63.5.1995-2001.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wahren B., Oberg B. Inhibition of cytomegalovirus late antigens by phosphonoformate. Intervirology. 1980;12(6):335–339. doi: 10.1159/000149093. [DOI] [PubMed] [Google Scholar]
  34. Wahren B., Wahren P., Harmenberg J., Sundqvist V. A. Computer-based virus sensitivity assay and neutralization method. Applications for herpesviruses. J Virol Methods. 1983 May;6(5):271–282. doi: 10.1016/0166-0934(83)90042-3. [DOI] [PubMed] [Google Scholar]
  35. Winston D. J., Ho W. G., Lin C. H., Bartoni K., Budinger M. D., Gale R. P., Champlin R. E. Intravenous immune globulin for prevention of cytomegalovirus infection and interstitial pneumonia after bone marrow transplantation. Ann Intern Med. 1987 Jan;106(1):12–18. doi: 10.7326/0003-4819-106-1-12. [DOI] [PubMed] [Google Scholar]

Articles from Clinical and Experimental Immunology are provided here courtesy of British Society for Immunology

RESOURCES