Skip to main content
Log in

Expression and purification of E2/NS1 protein of hepatitis C virus and detection of anti-E2/NS1 antibodies in chronic liver disease patients

  • Original Paper
  • Published:
Journal of Biomedical Science

Abstract

Glycoproteins on the surface of viral particles present the main target of neutralizing antibodies. The structural proteins of most Flaviviruses are known to elicit neutralizing antibodies and, thus, to help in both the natural resolution of the infection and the protection from challenge with homologous hepatitis C virus (HCV). Because such antigens are associated with the viral clearance in both humans and chimpanzees, we aimed to express the E2/NS1 protein of HCV and to study the role of anti-E2/NS1 antibodies in the natural resolution of HCV infection. The prevalence of anti-E2/NS1 antibodies to recombinant E2/NS1 protein was seen by Western blot in chronic liver disease patients (15 chronic hepatitis and 12 cirrhotic patients), who were positive for anti-HCV and negative for HBV infection. The study also included 2 negative controls (positive for HBV infection and negative for anti-HCV antibodies) and 2 healthy controls (negative for both HBV and HCV infection). Anti-E2/NS1 was present in 20% of the chronic hepatitis and 16% of the cirrhosis patients. None of the controls were positive for anti-E2/NS1 antibodies. Serum samples positive for anti-E2/NS1 antibodies were also positive for HCV RNA by RT/PCR. Accordingly, the presence of anti-E2/NS1 may have very little or no role in the natural resolution of HCV infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alter HJ, Margolis HS, Krawczynsky K, Judson FN, Mares A, Alexander WJ, Hu PY, Miller JK, Gerber MA, Sampliner RE, Meeks EL, Beach MJ. The natural history of community-acquired hepatitis C in United States. N Engl J Med 327:1899–1905;1992.

    Google Scholar 

  2. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H. Non-structural protein-3 of hepatitis C virus encodes a serine-type proteinase required for cleavage at NS3/NS4 and NS-4/NS5 junctions. J Virol 67:3835–3844;1993.

    Google Scholar 

  3. Bukh J, Purcell RH, Miller RH. At least 12 genotypes of hepatitis C virus predicted by sequence analysis of putative E1 gene of isolates collected worldwide. Proc Natl Acad Sci USA 90:8234–8238;1993.

    Google Scholar 

  4. Choo QL, Kuo G, Ralston R, Weiner A, Chien D, Van Nest G, Han J, Berger K, Thudium K, Kuo C, Kansopon J, McFarkland J, Tabrizi A, Ching K, Moss B, Cummins LB, Houghton M, Muchmore E. Vaccination of chimpanzees against infection by hepatitis C virus. Proc Natl Acad Sci USA 91:1294–1298;1994.

    Google Scholar 

  5. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362;1989.

    Google Scholar 

  6. Choo QL, Richman KH, Hans JH, Berger K, Lee C, Dong C, Gallegos C, Coit D, Selby R, Barr PJ. Genetic organization and diversity of hepatitis C virus. Proc Natl Acad Sci USA 6:2451–2455;1991.

    Google Scholar 

  7. Cocquerel L, Meunier, JC, Op de Beeck A, Bonte D, Wychowski C, Dubuisson J. Coexpression of hepatitis C virus envelope proteins E1 and E2 incis improves the stability of membrane insertion of E2. J Gen Virol 82:1629–1635;2001.

    Google Scholar 

  8. Duarte EA, Novella IS, Weaver SC, Domingo E, Wainhobson S, Clarke DK, Moya A, Elena SF, DelaTorre JC, Holland JJ. RNA virus quasispecies: Significance for viral disease and epidemiology. Infect Agents Dis 3:201–214;1994.

    Google Scholar 

  9. Dubuisson J, Duvet S, Meunier JC, Op De Beeck A, Cacan R, Wychowski C, Cocquerel L. Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. J Biol Chem 275:30605–30609;2000.

    Google Scholar 

  10. Emtage JS, Angal S, Doel MT, Harris TJR, Jenkins B, Lilley G, Lowe PA. Synthesis of calf prochymosin (prorenin) inEscherichia coli. Proc Natl Acad Sci USA 80:3671–3675;1983.

    Google Scholar 

  11. Farci P, Schimoda A, Wong D, Cabezon T, DeGioannis D, Strazzera A, Shimizu Y, Shapiro M, Alter HJ, Purchell RH. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of envelope 2 protein. Proc Natl Acad Sci USA 93:15394–15399;1996.

    Google Scholar 

  12. Grakoui A, Wychowski C, Lin C, Feinston SM, Rice CM. Expression and identification of HCV polyprotein cleavage products. J Virol 67:1385–1395;1993.

    Google Scholar 

  13. Houghton M, Weiner A, Hans J, Kuo G, Choo QL. Molecular biology of the hepatitis C viruses: Implication for diagnosis development and control of disease. Hepatology 14:381–388;1991.

    Google Scholar 

  14. Hussy P, Schmid G, Mous J, Jacobson H. Purification and in vitro phospholabeling of secretory envelope protein E1 and E2 of hepatitis C virus expressed in insect cells. Virus Res 45:45–57;1996.

    Google Scholar 

  15. Inchaupse G, Zebedee S, Lee DH, Sugitani M, Nasoff M, Prince AM. Genomic structure of the human prototype strain H of hepatitis C virus: Comparison with American & Japanese isolates. Proc Natl Acad Sci USA 88:10292–10296;1991.

    Google Scholar 

  16. Inove Y, Suzuki R, Matsuura Y, Harada S, Chiba J, Watanabe Y, Saito I, Miyamura T. Expression of the amino-terminal half of the NS1 region of the hepatitis C virus genome and detection of an antibody to the expressed protein in patients with liver disease. J Gen Virol 73:2151–2154;1992.

    Google Scholar 

  17. Kato N, Hijikata M, Ostsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients of non-A, non-B hepatitis. Proc Natl Acad Sci USA 87:9524–9528;1990.

    Google Scholar 

  18. Kato N, Dotsuyama Y, Janaka T, Nakagawa M, Nakazawa T, Muraiso K, Ohkoshi S, Hijikata M, Shimotohno K. Marked sequence diversity in the putative envelop proteins of hepatitis C virus. Virus Res 22:107–123;1992.

    Google Scholar 

  19. Kobayashi M, Tanaka E, Matsumoto A, Ichijo T, Kiyosawa K. Antibody response to E2/NS1 hepatitis C virus protein in patients with acute hepatitis C. J Gastroenterol Hepatol 12:73–76;1997.

    Google Scholar 

  20. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244:362–364;1989.

    Google Scholar 

  21. Lee JW, Kim KM, Jung SH, Lee KJ, Choi EC, Sung YC, Kang CY. Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein using mouse monoclonal antibodies. J Virol 73:11–18;1999.

    Google Scholar 

  22. Manabe S, Fuke I, Tanishita O, Kaji C, Gomi Y, Yoshida S, Mori C, Takamizawa A, Yosida I. Production of non-structural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology 198:636–644;1994.

    Google Scholar 

  23. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab, Cold Spring Harbor, 1989.

    Google Scholar 

  24. Marston FAO, Peter LA, Michael DT, Schoemaker JM, White S, Sarojani A. Purification of calf prochymosin (prorenin) synthesized inEscherichia coli. Biotechnology 2:800–305;1984.

    Google Scholar 

  25. Martell M, Esteban JI, Quer J, Genesca J, Weineer A, Esteban R, Guardia J, Gomez J. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: Quasispecies nature of HCV genome distribution. J Virol 66:3225–3229;1992.

    Google Scholar 

  26. Matsuura Y, Harada S, Suzuki R, Watanabe Y, Inoue Y, Saito I, Miyamura T. Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells. J Virol 66:1425–1431;1992.

    Google Scholar 

  27. Matsuura Y, Suzuki R, Suzuki T, Sato M, Azaki H, Saito I, Miyamura T. Processing of E1 and E2 glycoproteins of hepatitis C virus expressed in mammalian and insect cells. Virology 205:141–150;1994.

    Google Scholar 

  28. Merola M, Brazzoli M, Cocchiarella F, Heile JM, Helenius A, Weiner AJ, Houghton M, Abrignani S. Folding of hepatitis C virus E1 glycoprotein in a cell-free system. J Virol 75:11205–11217;2001.

    Google Scholar 

  29. Michalak JP, Wychowski C, Choukhi A, Meunier JC, Ung S, Rice CM, Dubuisson J. Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol 78:2299–2306;1997.

    Google Scholar 

  30. Prince AM, Brotman B, Lee DH, Ren L, Moore BS, Scheffel JW. Significance of the anti-E2 response in self-limited and chronic hepatitis C virus infections in chimpanzees and in humans. J Infect Dis 180:987–991;1999.

    Google Scholar 

  31. Ralston R, Thudium K, Berger K, Kuo C, Gervase B, Hall J, Selby M, Kuo G, Houghton M, Choo QL. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia virus. J Virol 67:6753–6761;1993.

    Google Scholar 

  32. Schlessinger JJ, Brandriss MW, Walsh EE. Protection against 17 D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the non-structural glycoprotein gp48 and by active immunization with gp48. J Immunol 135:2805–2809;1985.

    Google Scholar 

  33. Schlessinger JJ, Brandriss MW, Walsh EE. Protection of mice against dengue 2 virus encephalitis by immunization with dengue 2 virus non-structural glycoprotein NS-1. J Gen Virol 68:853–857;1987.

    Google Scholar 

  34. Shimotohno K. Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection. J Virol 68:4776–4784;1994.

    Google Scholar 

  35. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Onishi E, Andoh T, Yoshida I, Okayama H. Structure and organization of hepatitis C virus genome isolated from human carriers. J Virol 65:1105–1113;1991.

    Google Scholar 

  36. Tomei L, Failla C, Santolini E, DeFrancesco R, La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol 67:4017–4026;1993.

    Google Scholar 

  37. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354;1979.

    Google Scholar 

  38. Yokosuka O, Ito Y, Ohto M, Omata M. High detection rate of hepatitis C virus E2 antibody in patients with type C hepatitis. Gastroenterol Jpn 28(suppl)5:52–54;1993.

    Google Scholar 

  39. Yuki N, Hayashi N, Kasahara A, Hagiwara H, Mita E, Ohkawa K, Katayama K, Fusamoto H, Kamada T. Quantitative analysis of antibody to hepatitis C virus envelope2 glycoprotein in patients with chronic hepatitis C virus infection. Hepatology 23:947–952;1996.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pandya, J., Chakraborti, A. & Chawla, Y. Expression and purification of E2/NS1 protein of hepatitis C virus and detection of anti-E2/NS1 antibodies in chronic liver disease patients. J Biomed Sci 10, 276–282 (2003). https://doi.org/10.1007/BF02256063

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02256063

Key Words

Navigation