Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins L. V. Olenina,* L. I. Nikolaeva,* B. N. Sobolev,* N. P. Blokhina, A. I. Archakov,* and E. F. Kolesanova* *Institute of Biomedical Chemistry, Russian Academy of Medical Science, and  Specialized Hepatologic Center, 1st Moscow Infection Diseases Hospital, Moscow, Russia Received July 2001; accepted for publication November 2001 INTRODUCTION Hepatitis C virus (HCV) classified in the family Flaviviridae is a major ethiological agent of the parenterally transmitted non-A non-B hepatitis world-wide. Up to 80% of HCV infections lead to chronic persistence of the virus that may lead to cirrhosis and hepatocellular carcinoma [1]. No prophylactic vaccine against HCV is available. The high frequency of the development of chronic infec- tion confirms the inefficiency of the host immune system. Mechanisms leading to HCV persistence are not clearly understood. However, they evidently include the existence of immunoprivileged sites of viral replication, the emergence of immune escape variants [2,3] and the masking of part of the viral surface by lipoprotein molecules [4]. HCV is a small positive-stranded RNA virus with at least six genotypes, some of which are divided further into several subtypes [5]. Moreover, inside each patient HCV exists as a set of genetically different but closely related variants, the so-- called quasispecies [6]. This set can be modulated by the host immune pressure. The HCV genome contains a single open reading frame (ORF) that codes a precursor polyprotein which is cotranslationally and post-translationally processed into three structural (core and two envelope glycoproteins, E1 and E2) and six nonstructural proteins (NS2-NS5). The exposure of the HCV envelope glycoproteins on the virion surface makes them targets for antibodies that may block key functions of these glycoproteins and prevent viral entry into the host cell. E1 and E2 are typical viral glycopro- teins (about 190 and 380 amino acid residues, respectively), which contain putative transmembrane regions represented by hydrophobic residue clusters at their C-termini. These proteins are highly glycosylated and show great variability among viral isolates, with E2 N-terminal sequence (27 amino acid residues) known as the most variable site, called hyper- variable region 1 (HVR1). It has been shown that only half of E2 amino acid positions and one third of E1 are highly con- servative [7]. E1 and E2 outer domains form noncovalently bound heterodimers on the membrane surface [8]. Chimpanzees immunized with purified recombinant E1/E2 heterodimers have been protected partly against rechallenge with homologous isolates of virus [9]. This protection is correlated with the presence of antibodies capable of blocking viral replication. These data support the importance of HCV envelope proteins in eliciting virus-neutralizing immune responses. However, several studies show that virus-neutralizing antibodies are raised against HVR1. Participation of antiHVR1 antibodies in virus neutralization has been Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus; HVR1, hypervariable region 1; ORF, open reading frame; PBS, phosphate buffered saline. Correspondence: Ludmila V. Olenina, Institute of Biomedical Chemistry, Russian Academy of Medical Science, Moscow, Russia. E-mail: oleninal@ibmh.msk.su Journal of Viral Hepatitis, 2002, 9, 174–182 Ó 2002 Blackwell Science Ltd SUMMARY. Forty-eight overlapping octapeptides covering highly conservative regions of E1 and E2 hepatitis C virus (HCV) envelope proteins were synthesized and tested by ELISA against different groups of sera obtained from HCV- infected patients. All sera from patients with acute infection, except a single case of serum reactivity with the region HINRTALN, were nonreactive with any peptide. Sera ob- tained from chronic patients reacted with 12 peptides from five selected regions. Two immunodominant B epitopes were found, one being the precisely mapped antigenic site RMAWDM positioned inside the earlier shown immuno- dominant epitope from E1, and the second site, PALSTGLIH from E2, detected for the first time. New minor antigenic site was determined as PTDCFRKH from E2. We found only minor seroreactivity for one of the putative sites involved in CD81 binding, PYCWHYAP. Keywords: HCV, envelope, epitope mapping, pepscan.