Hepatitis C Virus E2 Protein Purified from Mammalian Cells Is Frequently Recognized by E2-specific Antibodies in Patient Sera

Abstract

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by β-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s). The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by β-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s). Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; hGH, human growth hormone; CHO, chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; IRES, internal ribosome entry sequence; EMCV, encephalomyocarditis virus; PCR, polymerase chain reaction; DHFR, dihydrofolate reductase; PBS, phosphate-buffered saline; mAb, monoclonal antibody; β-ME, β-mercaptoethanol. is a major causative agent of post-transfusion and sporadic non-A, non-B hepatitis throughout the world (1Choo Q.-L. Kuo G. Weiner A. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6292) Google Scholar, 2Kuo G. Choo Q.-L. Alter H.J. Gitnick G.L. Redeker A.G. Purcell R.H. Miyamura T. Dienstag J.L. Alter M.J. Stevens C.E. Tegtmeier G.E. Bonino F. Colombo M. Lee W.-S. Kuo C. Berger K. Shuster J.R. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 362-364Crossref PubMed Scopus (3059) Google Scholar). In most cases, the virus appears to cause a persistent infection. Previous studies indicate that the development of chronic liver diseases, cirrhosis, and hepatocellular carcinoma is associated with chronic HCV infection (3Saito I. Miyamura T. Ohbayashi A. Harada H. Katayama T. KiKuchi S. Watanabe Y. Koi S. Onji M. Ohta Y. Choo Q.-L. Houghton M. Kuo G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6547-6549Crossref PubMed Scopus (1085) Google Scholar). Comparative analyses of the genomes from several HCV strains indicate that HCV is a member of the family Flaviviridae, which includes flaviviruses and pestiviruses (4Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby A. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-2455Crossref PubMed Scopus (1531) Google Scholar). The HCV genome is a 9.5-kilobase positive-strand RNA from which a single polypeptide is expressed and processed by cellular and viral proteinases to produce the putative viral structural and nonstructural proteins (4Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby A. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-2455Crossref PubMed Scopus (1531) Google Scholar, 5Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 6Selby M.J. Choo Q.-L. Berger K. Kuo G. Glazer E. Eckart M. Lee C. Chien D. Kuo C. Houghton M. J. Gen. Virol. 1993; 74: 1103-1113Crossref PubMed Scopus (200) Google Scholar). It was previously shown that structural proteins were composed of the core protein of 18–22 kDa and two glycosylated envelope proteins, E1 of 31–35 kDa and E2 of 58–74 kDa (5Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 7Fournillier Jacob A. Cahour A. Escriou N. Girad M. Wychowski C. J. Gen. Virol. 1996; 77: 1055-1064Crossref PubMed Scopus (43) Google Scholar, 8Harada S. Suzuki R. Ando A. Watanabe Y. Yagi S. Miyamura T. Saito I. J. Gen. Virol. 1995; 76: 1223-1231Crossref PubMed Scopus (30) Google Scholar, 9Lanford R.E. Notvall L. Chavez D. White R. Frenzel G. Simonsen C. Kim J. Virology. 1993; 197: 225-235Crossref PubMed Scopus (104) Google Scholar, 10Matsuura Y. Suzuki T. Suzuki R. Sato M. Aiziki H. Saito I. Miyaramura T. Virology. 1994; 205: 141-150Crossref PubMed Scopus (86) Google Scholar, 11Nishihara T. Nozaki C. Nakatake H. Hoshiko K. Esumi M. Hayashi N. Hino K. Hamada F. Mizuno K. Shikata T. Gene (Amst.). 1993; 129: 207-214Crossref PubMed Scopus (20) Google Scholar). Although some lymphocyte cell lines have shown to support the limited replication of HCV, there has not been in vitro cell culture system efficiently enough to be used for viral propagation and for detailed virological studies (12Shimizu Y.K. Iwamoto A. Huikata M. Purcell R.H. Yoshikura H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5477-5481Crossref PubMed Scopus (319) Google Scholar). Expression studies using recombinant cDNA templates are the only means for identifying individual HCV proteins and to study their roles in the pathogenesis of HCV infection. The hydrophobicity profile of HCV polyprotein suggested that the HCV E2 protein corresponds to the flavivirus NS1 glycoprotein and the major pestivirus envelope protein gp53/gp55 (E2; gp53 in bovine viral diarrhea virus and gp55 in hog cholera virus), which were reported to induce protective immunity in experimental animals (13Hulst M.M. Westra D.F. Wensvoort G. Moormann R.J.M. J. Virol. 1993; 67: 5435-5442Crossref PubMed Google Scholar, 14Rumenapf T. Unger G. Strauss J.H. Thiel H.-J. J. Virol. 1993; 67: 3288-3294Crossref PubMed Google Scholar). HCV envelope proteins are of considerable interest, because experimentally challenged chimpanzees were either protected or shown to ameliorate disease following vaccination with recombinant E1/E2 subunits (15Choo Q.-L. Kuo G. Ralston R. Weiner A. Chien D. Van Nest G. Han J. Berger K. Thudium K. Kuo C. Kansopon J. McFarland J. Tabrizi A. Ching K. Moss B. Cummins L.B. Houghton M. Muchmore E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1294-1298Crossref PubMed Scopus (537) Google Scholar). It was recently reported that HCV E2 protein expressed in Chinese hamster ovary (CHO) cells binds to target cells at a high affinity. In addition, antibody which neutralizes the binding of E2 to target cell appears to correlate with protection from HCV infection (16Rosa D. Campagnoli S. Moretto C. Guenzi E. Cousens L. Chin M. Dong C. Weiner A.J. Lau J.Y. Choo Q.-L. Chien D. Pikeri P. Houghton M. Abrignani S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1759-1763Crossref PubMed Scopus (323) Google Scholar). These results suggest that HCV E2 protein is a key viral antigen for a hepatitis C vaccine. The HCV envelope protein expressed in cells infected with recombinant baculovirus and vaccinia virus was used for detection of envelope-specific antibody in patient sera (17Chien D.Y. Choo Q.-L. Ralston R. Spaete R. Tong M. Houghton M. Kuo G. Lancet. 1993; 342: 933Abstract PubMed Scopus (86) Google Scholar, 18Hsu H.H. Donets M. Greenberg H.B. Feinstone S.M. Hepatology. 1993; 17: 763-771PubMed Google Scholar, 19Inoue Y. Suzuki R. Matsuura Y. Harada S. Chiba J. Watanabe Y. Saito I. Miyamura T. J. Gen. Virol. 1992; 73: 2151-2154Crossref PubMed Scopus (32) Google Scholar). However, the purification of HCV envelope protein at a high yield was thought to be a difficult task. When HCV envelope proteins were purified from HeLa cells infected with recombinant vaccinia virus, approximately 1.5 of purified E1/E2 protein was from a culture of infected HeLa cells (15Choo Q.-L. Kuo G. Ralston R. Weiner A. Chien D. Van Nest G. Han J. Berger K. Thudium K. Kuo C. Kansopon J. McFarland J. Tabrizi A. Ching K. Moss B. Cummins L.B. Houghton M. Muchmore E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1294-1298Crossref PubMed Scopus (537) Google Scholar). the yield of purified E1/E2 protein appeared to be to the of the HCV envelope and studies with E1 and E2 protein has been In we two recombinant cell lines expressing a and a secretory hGHE2t fusion of human growth hormone (hGH), and a C-terminal truncated E2 (E2t) The fusion protein was shown to be a and and purified to 80% by using chromatography from the culture of antibody in sera and antigenic of the purified hGHE2t protein were by enzyme-linked immunosorbent were from and to and in from to were from were with high and for antibody HCV includes recombinant of core and purified from cells and of and When HCV was with HCV using the results of HCV with that of HCV results were by using and HCV of was shown to be with HCV two were with HCV The is The of chronic hepatitis and liver was by persistent liver for and for of and/or liver the of hepatocellular and were in HCV which at in for were as chronic from with chronic were were a we used from and were for HCV HCV cDNA E1 and E2 was by of RNA from a of HCV-positive patient Kim C.M. 1993; Scholar). The cDNA was from and the of a The of the was and into To internal ribosome entry of encephalomyocarditis virus was by from B. T. 1990; PubMed Scopus Google using and and the was into of was constructed by the of into of 1989; PubMed Scopus Google Scholar). The and of HCV polyprotein were by using and or The with and were into of of and the fusion were with and into to and was constructed by the cDNA of into PubMed Scopus Google Scholar). To the the of was by from 1991; PubMed Scopus Google using and The was in fused to of of E2 and were by using and or and were in fused to the of to produce and The to be from the HCV envelope protein purification of fusion of and hGHE2t fusion proteins was by in cells were in and cells in dihydrofolate were in and and cells were with the using C. H. PubMed Scopus Google Scholar). cells were as previously in C. L. Van Nest G. F. D. J. Virol. PubMed Google Scholar). cell lines expressing recombinant HCV E2 protein were using and with from HCV patient and anti-hGH antibody and were to of of a patient with chronic hepatitis C was as a by using HCV anti-hGH antibody was the purified protein expressed in antibody and with were from mAb was purified from a from the cells were and with as previously R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar). The cells were with and by centrifugation at for of and culture supernatant were either with from HCV-positive patient or anti-hGH were by using as previously E. D. Scholar). were and by SDS-polyacrylamide gel was to the as previously R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar). of proteins a were with the in for at from HCV-positive patient or anti-hGH antibody in as the antibody and either or with in as the antibody were used to HCV E2 protein and by The of and intramolecular disulfide was by the of and PubMed Scopus Google Scholar). of HCV proteins was by The purified hGH, the hGHE2t fusion protein in and in were of density and was at for in a at were by the of the and of were by were with either the culture supernatant from cells or the purified hGHE2t protein of the purified protein with SDS, with or at for and with The were with bovine and by with sera from HCV-positive at for of the and of the were with with The was by the of mAb purified from by was to of to the When cells expressing hGHE2t protein were to culture was with the was and to the mAb affinity with The was with and in PBS, and the hGHE2t protein was with in The was the purification of C-terminal truncated E2 the purified hGHE2t fusion protein was and with for and to mAb affinity The C-terminal truncated E2 protein was purified by the The purified hGHE2t protein was to and β-ME, and for Treatment of was in the SDS, and for at HCV RNA was from by as previously H. S. Y. K. H. A. Miyamura Y. M. J. Gen. Virol. 1991; Scopus Google Scholar). and using and a were as previously Kim C.M. 1993; and were by electrophoresis and studies of HCV envelope proteins in have been limited to a of in vitro cell culture system virus In addition, the purification of a HCV envelope protein appears to be difficult because HCV envelope proteins are To and HCV E2 protein at a high level, we several for a recombinant cell and were to the HCV E2 with and a C-terminal the of E2 was with the of hGH, because was that the of E2 was not for the and that was at a high and efficiently into culture in J. Y. G. and we two fusion and of the E2 of in which the was of E2 to the from the fusion protein To the were of expressing the recombinant were by the in shown in and proteins were expressed as of and when cell were with sera from HCV-positive patient and In specific protein were not in cell with either or as a and The of hGHE2t and fusion proteins by sera from HCV-positive patient were when the was with anti-hGH antibody not These results indicate that and proteins expressed in cells were with antibodies in To the expressed E2 protein be the culture supernatant of cells was with sera from HCV-positive E2 protein was in culture of cells with and In a of hGHE2t and proteins with of and were in the culture supernatant and It is that the hGHE2t protein is efficiently the These results indicate that E2 proteins, with and a C-terminal E2 and are of into culture when secretory as hGH, was fused to the E2 The form appeared to be its to the of the secretory the of we for the of a recombinant cell line expressing E2 as a secretory and as a were into and cell lines were using and with anti-hGH of the recombinant and cell lines were at a to and of the of hGHE2t and protein was shown to be and by anti-hGH The hGHE2t protein as as the of kDa by sera from HCV-positive patient in not These results suggest that the hGHE2t protein in cells as as cells is a secretory protein which is To hGHE2t fusion the mAb affinity was by the mAb to the of recombinant and cells were to the mAb affinity and with and in PBS, and hGHE2t protein was with chromatography approximately of hGHE2t protein with 80% were from of culture of the recombinant The hGHE2t protein purified from recombinant cell line a of kDa gel with and was with sera from HCV-positive patient and The protein was by mAb affinity The purified protein a and a in SDS-polyacrylamide gel electrophoresis of the in and at the The the of are in which and indicate the and of is that and at the are from the at of the purified hGHE2t fusion It is that the and are by of at The hGHE2t fusion protein was used to the HCV E2 protein for the following the fusion protein appears to be by at in a E2 the binding of the E2 protein to antibody was shown to be to β-ME, which be in the of Although was reported that E1 and E2 protein form a is that is by either intermolecular disulfide bonds and/or Y. Suzuki T. Suzuki R. Sato M. Aiziki H. Saito I. Miyaramura T. Virology. 1994; 205: 141-150Crossref PubMed Scopus (86) Google Scholar, R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar, M.J. Glazer E. F. Houghton M. Virology. 1994; PubMed Scopus Google Scholar). In addition, a of E1 or E2 protein to be we the intermolecular of purified hGHE2t fusion protein by the of and PubMed Scopus Google which disulfide by by of from reduced into high were These high in to reduced and as as and that were by the of disulfide bond(s) To of the hGHE2t the hGHE2t fusion and the proteins were to by that the hGHE2t fusion but not the purified protein not as and results suggest that the HCV E2 protein as a form appears to be a of and oligomers as and To the of antibody in chronic non-A, non-B hepatitis and in sera were by using the hGHE2t protein The sera was with the protein in and with with It was that anti-hGH antibody was not in when the protein purified from cell as a was in not The hGHE2t protein was shown to with antibodies in and of sera from with chronic liver cirrhosis, and hepatocellular of sera from and with a high appeared to with the hGHE2t These results suggest that antibody is in 90% of sera from of antibody in sera using the purified hGHE2t of patient of of for of was by using HCV with a high were to be for antibody non-B with and for antibody antibody with chronic and antibody in sera was by enzyme-linked immunosorbent with the recombinant purified hGHE2t protein antibody was not in sera when purified protein was used as a the of the the of sera to be for for of was by using HCV with a high were to be for antibody non-B with and for antibody with chronic and in a antibody in sera was by enzyme-linked immunosorbent with the recombinant purified hGHE2t protein antibody was not in sera when purified protein was used as a the of the the of sera to be for The of antibody in with chronic was by using the hGHE2t of from are shown to be by HCV and using the hGHE2t sera, which were with the hGHE2t protein To were HCV HCV RNA was and by with and of HCV by HCV These results indicate that E2 protein is to be in the of HCV infection for with chronic The major antigenic of HCV envelope proteins is thought to be because the E2 protein is glycosylated and recognized in a form in a form by sera from HCV-positive S. Suzuki R. Ando A. Watanabe Y. Yagi S. Miyamura T. Saito I. J. Gen. Virol. 1995; 76: 1223-1231Crossref PubMed Scopus (30) Google Scholar). To the of antigenic the hGHE2t fusion protein was with and/or and by using HCV-positive SDS, and were used at that not The protein with either or for appeared to have the binding to sera as efficiently as protein In with either or reactivity in 80% of These results suggest that intermolecular and/or intramolecular disulfide of hGHE2t protein are important for the of the antigenic of sera to the purified hGHE2t protein from with of antibody were for the reactivity to hGHE2t protein at The purified hGHE2t protein was with heat by in the of and with The hGHE2t protein was into and was as The the of density at in a from with of antibody were for the reactivity to hGHE2t protein at The purified hGHE2t protein was with heat by in the of and with The hGHE2t protein was into and was as The the of density at When with the hGHE2t protein was shown to be glycoprotein not which is with R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar). To the of in the binding of E2 purified protein was with by F. not the binding ability of the hGHE2t protein to suggesting that the of the E2 protein is not for the binding of the E2 protein to its specific In we a recombinant cell line expressing HCV E2 as a secretory The hGHE2t fusion protein was at a high and purified to 80% using chromatography The purified hGHE2t protein was recognized by sera from 90% of HCV-positive and of patients. of the purified protein to antibody from sera appeared to be reduced by the with but not with as and The hydrophobicity profile of HCV polyprotein that the C-terminal of the E2 protein is (4Choo Q.-L. Richman K.H. Han J.H. Berger K. Lee C. Dong C. Gallegos C. Coit D. Medina-Selby A. Barr P.J. Weiner A.J. Bradley D.W. Kuo G. Houghton M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2451-2455Crossref PubMed Scopus (1531) Google Scholar). It was previously reported that the E2 protein to the putative and that of the C-terminal appeared to the of the truncated E2 protein Y. Suzuki T. Suzuki R. Sato M. Aiziki H. Saito I. Miyaramura T. Virology. 1994; 205: 141-150Crossref PubMed Scopus (86) Google Scholar, R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar, M.J. Glazer E. F. Houghton M. Virology. 1994; PubMed Scopus Google D. Choo Q.-L. Berger K. K. Kuo C. S. Lee C. Ralston R. Thudium K. Kuo G. Houghton M. Virology. 1992; PubMed Scopus Google Scholar). The of proteins as and virus glycoprotein was shown to the of the E2 but not the E2 protein T. Nozaki C. Nakatake H. Hoshiko K. Esumi M. Hayashi N. Hino K. Hamada F. Mizuno K. Shikata T. Gene (Amst.). 1993; 129: 207-214Crossref PubMed Scopus (20) Google Scholar, D. Choo Q.-L. Berger K. K. Kuo C. S. Lee C. Ralston R. Thudium K. Kuo G. Houghton M. Virology. 1992; PubMed Scopus Google Scholar). In that the fusion of to the E2 protein E2 protein as as the form to efficiently into culture suggesting that HCV E2 protein be the and/or secretory protein fused to the E2 The major glycoprotein of pestiviruses was shown to form and and and (13Hulst M.M. Westra D.F. Wensvoort G. Moormann R.J.M. J. Virol. 1993; 67: 5435-5442Crossref PubMed Google Scholar, E. R. B. T. G. Thiel H.-J. J. Virol. 1990; PubMed Google Scholar). The of HCV E1 and E2 is a of It was reported that a of E1 and E2 in of cells infected with recombinant vaccinia virus was associated disulfide R. Thudium K. Berger K. Kuo C. B. J. M. Kuo G. Houghton M. Choo Q.-L. J. Virol. 1993; 67: PubMed Google Scholar). In that E1 and E2 proteins expressed in and HeLa cells are associated (5Grakoui A. Wychowski C. Lin C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 1385-1395Crossref PubMed Google Scholar, 10Matsuura Y. Suzuki T. Suzuki R. Sato M. Aiziki H. Saito I. Miyaramura T. Virology. 1994; 205: 141-150Crossref PubMed Scopus (86) Google Scholar). results that the hGHE2t protein appeared to form a and which are linked by intermolecular disulfide is to be of E2 because was not in of envelope protein in RNA as virus protein and and human virus envelope has been S. T. G. Moss B. Virology. 1990; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. G. J. Gen. Virol. 1994; PubMed Scopus Google Scholar). It was reported that was shown to be for and cell of viral A. Virology. 1993; PubMed Scopus Google and that the and the protein of virus are as in virus, the protein of virus has a to into a G. G. J. Virol. 1989; PubMed Google Scholar). Although the of E1 and E2 proteins in the is not support that the and of envelope proteins of HCV be to of in which the of envelope proteins, and is by intermolecular disulfide When antibody of patient sera were by using the purified hGHE2t a of antibody were in infected to not is with the results (15Choo Q.-L. Kuo G. Ralston R. Weiner A. Chien D. Van Nest G. Han J. Berger K. Thudium K. Kuo C. Kansopon J. McFarland J. Tabrizi A. Ching K. Moss B. Cummins L.B. Houghton M. Muchmore E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1294-1298Crossref PubMed Scopus (537) Google Scholar). However, the the of antibody and specific liver disease was not It is to that several of sera were as with antibody detection and using HCV was shown to be be in as with chronic is that the antibody to protein is not enough to be by the In the antibody to envelope protein be because the E2 glycoprotein the of the infected cell and the be by cells and/or as a antigen to its In the E2 protein of HCV has the to be used for the detection of HCV infection in patients. It was previously reported that antibodies from with hepatitis C were to bind to the E2 protein to the E2 protein Y. Suzuki T. Suzuki R. Sato M. Aiziki H. Saito I. Miyaramura T. Virology. 1994; 205: 141-150Crossref PubMed Scopus (86) Google Scholar). In addition, monoclonal antibodies the envelope protein of hog cholera virus appeared to only with its suggesting that antibodies to recognize discontinuous of which be by the of G. G. J. Virol. 1989; PubMed Google Scholar). In purified hGHE2t protein with or not in the reactivity to antibody of The be to the and/or the E2 protein The reactivity of the purified hGHE2t protein to antibody by of a suggesting that the purified hGHE2t protein has a discontinuous antigenic by disulfide