B-cell epitopes in hypervariable region 1 of hepatitis C virus obtained from patients with chronic persistent hepatitis

The hypervariable domain (HVR1) within the N-terminus of the E2 protein of hepatitis C virus (HCV) is known to be variable antigenically during the course of persistent infection. The aim of the study was to detect B-cell epitopes in HVR1 responsible for neutralizing HCV. The B-cell epitopes were analyzed using two series of synthetic peptides: 25 heptapeptides from the most common amino acids within 73 HVR1 sequences, and 216 heptapeptides, the sequences of which cover more than 65% of the 73 HVR1 sequences. Sera from three patients with chronic hepatitis C were tested for reactivity to the synthetic peptide sequences by enzyme-linked immunosorbent assay (ELISA). The post-interferon (IFN) serum of one patient who had a long-term response to treatment reacted specifically with 13 heptapeptides of 216 variable sequences of HVR1. Some of the amino acid sequences (amino acids 398, 399, 400, 404) of the heptapeptides were also found in those deduced from the nucleotide sequences of HCV genomes in the pre-IFN serum. The sera of the other two patients who did not respond to treatment did not react with the 13 heptapeptides. It is concluded that the B-cell epitopes in HVR1 may be relevant for eliminating viremia in the case of the patient who had a good response to treatment. These results suggest that the analysis of the B-cell epitopes recognized in HVR1 may be important in understanding the mechanism of persistent infection and progression of hepatitis. © 1996 Wiley-Liss, Inc.     

The hypervariable region 1 protein of hepatitis C virus broadly reactive with sera of patients with chronic hepatitis C has a similar amino acid sequence with the consensus sequence.

Hypervariable region 1 (HVR1) proteins of hepatitis C virus (HCV) have been reported to react broadly with sera of patients with HCV infection. However, the variability of the broad reactivity of individual HVR1 proteins has not been elucidated. We assessed the reactivity of 25 different HVR1 proteins (genotype 1b) with sera of 81 patients with HCV infection (genotype 1b) by Western blot. HVR1 proteins reacted with 2-60 sera. The number of sera reactive with each HVR1 protein significantly correlated with the number of amino acid residues identical to the consensus sequence defined by Puntoriero et al. (G. Puntoriero, A. Lahm, S. Zucchelli, B. B. Ercole, R. Tafi, M. Penzzanera, M. U. Mondelli, R. Cortese, A. Tramontano, G. Galfre', and A. Nicosia. 1998. EMBO J. 17, 3521-3533. ) (r = 0.561, P < 0.005). The most widely reactive HVR1 protein, 12-22, had a sequence similar to the consensus sequence. The peptide with C-terminal 13-amino-acids sequence of HVR1 protein 12-22 (NH2-CSFTSLFTPGPSQK) was injected into rabbits as an immunogen. The rabbit immune sera reacted with 9 of 25 HVR1 proteins of genotype 1b including HVR1 protein 12-22 and with 3 of 12 proteins of genotype 2a. These results indicate that the HVR1 protein broadly reactive with patients' sera has a sequence similar to the consensus sequence, can induce broadly reactive sera, and could be one of the candidate immunogens in a prophylactic vaccine against HCV.     

Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection

Summary. Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure.In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.     

Sequence evolution and cross-reactive antibody responses to hypervariable region 1 in acute hepatitis C virus infection

Hepatitis C virus (HCV) infection may result in acute resolving or chronic infection. Patients that clear the infection have a more vigorous cellular immune response and an early humoral response to the hypervariable region 1 (HVR1) of the E2 envelope protein. To analyse further the properties of the early anti-HVR1 response, cross-reactivity of anti-HVR1 responses was assessed in five patients with acute HCV infection, who were infected by the same virus strain during a nosocomial outbreak. The sequence evolution of HVR1 was examined in sequential serum samples up to 37 months post infection. Peptides were synthesised corresponding to the obtained HVR1 sequences and unrelated HVR1 sequences, and antibody reactivity to the peptides in sequential sera was investigated by ELISA. The results suggest an association between specific gaps in humoral immunity and the HVR1 sequence evolution during early infection. Possible interpretations of this phenomenon include immune escape mechanisms or suppression of specific anti-HVR1 antibodies.     

Development of a plant-derived subunit vaccine candidate against hepatitis C virus

Summary.  Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 “mimotopes” previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines. Accepted June 2, 2000 Received March 27, 2000     

Sequential change of the hypervariable region of the hepatitis C virus genome in acute infection

Hepatitis C virus (HCV) infection is characterized by persistence of liver inflammation that often leads to end-stage liver disease, although the mechanisms are not fully understood. A hyper-variable region (HVR) has been reported in the E2/NS1 region of the HCV genome, in which striking diversity is found among different HCV isolates. To investigate the association of the HVR alterations with the clinical courses of HCV infection, a longitudinal analysis of the HVR in patients with acute HCV infection was carried out. Plasma samples were obtained at several times in three patients with acute hepatitis C. Plasma RNA was extracted and reverse transcribed, and DNA fragments that included the HVR were amplified by PCR. The sequences of the HVR were directly determined from the PCR products by the dideoxy chain termination method, from which amino acid sequences were deduced. In all cases, plasma HCV-RNA disappeared with the improvement of the initial alanine aminotransferase (ALT) elevation, but HCV-RNA reappeared about 1 year later with or without deterioration of the hepatitis. In a case of sporadic acute hepatitis, the HCV in the recurrent phase had seven amino acid substitutions in the HVR compared with that in the acute phase, although no amino acid changes were noted during the initial acute phase. In a case of posttransfusion hepatitis, a marked difference was observed between the acute and the recurrent phases, with an amino acid homology of 30% (8/27). The mutation rate of the HVR had a tendency to accelerate as the HCV infection progressed to the chronic stage. In conclusion, the HVR changes serially during the course of acute HCV infection, and these HVR changes may play a part in the chronicity of HCV infection. © 1994 Wiiey-Liss, Inc.     

Hypervariable region diversity of hepatitis C virus and humoral response: comparison between patients with or without cirrhosis.

To investigate the potential clinical utility of antibody response to HVR1 of HCV, the genomic and amino acid diversity of HVR1 was compared between two groups of four chronic HCV carriers with or without liver cirrhosis. Peptides corresponding to the deduced COOH- and NH2-terminal amino acid sequences of HVR1 were synthesised to assess the reactivity of patient sera to autologous and homologous HVR1 epitopes by enzyme-linked immunosorbent assay. HCV chronic carriers had significantly more frequent cross-reactivity with homologous C- than N-terminal HVR1 peptides. Twelve cirrhotic and eleven noncirrhotic patients had a similar frequency of cross-reactivity with either C- or N-terminal HVR1 peptides. However, noncirrhotic patients had a significantly higher level of C-terminal HVR1 antibody cross-reactivity than cirrhotic patients. In HCV chronic carriers, the magnitude of the immune response to but not the frequency of cross-reactivity with C-terminus HVR1 peptides differ between patients with and without liver cirrhosis.     

Marked sequence diversity in the putative envelope proteins of hepatitis C viruses.

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)     

A highly hydrophobic domain within hypervariable region 1 may be related to the entry of hepatitis C virus into cultured human hepatoma cells

Interaction of the envelope glycoprotein of hepatitis C virus (HCV) with a cellular receptor(s) is thought to be essential for the initial steps of HCV infection. However, the mechanisms of HCV infection remain unclear. The aim of the present study was to determine the features of HCV that enable efficient entry of the virus into human hepatocytes. Variations of hypervariable region 1 (HVR1) sequences in HCV inocula and in infected human hepatoblastoma HepG2 cells were examined. Immunofluorescence of inoculated HepG2 cells with anti-HCV core antibodies demonstrated that HCV structural proteins were expressed in the cytoplasm, and their entry into HepG2 cells was confirmed. When the HVR1 amino acid sequences were compared, HVR1 quasispecies in the inoculated cells showed more uniformity than those of the inocula. Although there were no statistically significant differences between the two groups, hydrophobic residues were observed more frequently in the HVR1 amino acids from inoculated cells than in the HVR1 amino acids from the inocula. Results of hydropathy analysis revealed that highly hydrophobic domains exist in the middle of HVR1 in the inoculated cells in 7 of 10 patients. The results suggest that limited HCV populations are able to enter HepG2 cells and that the highly hydrophobic domain existing within the HVR1 may play an important role in the entry of HCV into cells.     

Predominance of antibodies to hepatitis C virus envelope proteins in various disease statuses of hepatitis C

The antibody profile to various proteins of hepatitis C virus (HCV) was studied in 113 patients positive for HCV RNA in various disease statuses of hepatitis C (HC). A single peptide (E2/NS1, aa 413-436 of HCV polyprotein) chosen from a conserved region at the C-terminus of the hypervariable region (HVR) HVR1 of HCV was found to be sufficient for reliable diagnosis of the infection, even in the acute phase. Six hundred and one suspected HC cases and 200 voluntary blood donors were tested by this peptide. The sensitivity of detection of HCV antibodies by this peptide did not increase with addition of peptides from other HCV proteins. Our results clearly demonstrate that antibodies to HCV envelope proteins occur in a higher percentage of the infected population than those to other proteins. This emphasizes the necessity of using representative sequences from HCV envelope proteins in diagnostic immunoassays of this viral infection.     

Broadly cross-reactive, high-affinity antibody to hypervariable region 1 of the hepatitis C virus in rabbits.

The HCV hypervariable region 1 (HVR1) of the main E2 envelope protein is critically important in HCV neutralization but its extreme variability makes immune therapy and vaccine development particularly difficult. To explore the hypothesis that HVR1 carries a common epitope susceptible of eliciting cross-reactive neutralizing and inhibitory antibodies, rabbits were immunized with a series of synthetic HVR1 peptides. The anti-HVR1 produced were purified and characterized. Several lines of evidence supported the working hypothesis: (1) although injected only once, a boosting effect from poorly homologous peptides was observed; (2) purified rabbit IgG reacted with high affinity with immunizing peptides and cross-reacted with 16 of 17 unrelated HVR1 peptides; (3) antibodies appeared of restricted diversity irrespective of the linear HVR1 peptide sequences; (4) anti-HVR1 peptides effectively captured HCV in 22 of 33 plasmas from random infected patients; (5) anti-HVR1 IgG blocked the binding of antibody-captured HCV to MOLT-4 cells. These findings suggest that with an appropriate HVR1 peptide immunization scheme, high titer, broadly cross-reactive, blocking antibodies to HCV can be produced. Antibodies to the putative ubiquitous HVR1 epitope may have important clinical uses.     

Genetic characterization of hypervariable region 1 in patients chronically infected with hepatitis C virus genotype 2.

The hypervariable region 1 (HVR1) has been most reliably identified in the genome of HCV genotype 1 isolates and thought to possibly play a role in immune evasion and development of chronic infection. There are few studies, however, of other HCV genotypes to determine if they also have such a hypervariable region present, and it is unclear whether or not there is any genotype-dependent difference in the genetic characteristics of HVR1. We determined the nucleotide sequence of 5' end of E2/NS1 region of the HCV genome spanning HVR1 of multiple genotype 1 and 2 HCV isolates and carried out a detailed genetic analysis. Similarity plots identified two hypervariable regions within the genotype 2 sequences, a larger one corresponding to HVR1 as well as a smaller 27-nucleotide region of hypervariability. The synonymous substitutions per synonymous site (ds) was greater than nonsynonymous substitutions per nonsynonymous site (dn) within genotype 1 group whereas dn and ds were similar in the genotype 2 group. Analysis of amino acid sequences revealed several conserved sites across genotype 1 and 2 (amino acid numbers 2,6, 20 and 26) and overall similar hydropathic profiles were found within two genotypes. Still, despite the hypervariability, the HVR1 showed a genotype-specific phylogenetic clustering. Thus, HVR1 appears to be conserved between genotypes in keeping with it having an important survival function. Genotype 2 appears to have a greater rate of nonsynonymous substitutions within HVR1, suggesting a greater positive evolutionary pressure.     

丙型肝炎病毒E2／NS1区基因cDNA的克隆及序列分析

从北京地区１份丙型肝炎病毒（ＨＣＶ）感染者血清中提取ＲＮＡ，经逆转录和套式聚合酶链反应扩增ＨＣＶＥ２／ＮＳ１区基因约９３０ｂｐ，将其插入至ｐＧＥＭ－Ｔ质粒载体中，利用双脱氧链末端终止法测出该基因５’端４３１ｂｐ的核苷酸序列，将此序列与其它９个ＨＣＶ分离株的相应序列进行比较分析，结果表明，此序列与ＨＣＶⅡ型序列同源性较高，核苷酸水平同源性在８０％以上。由其编码的ＨＣＶ外膜蛋白Ｎ端存在两个高变部位（ＨＶＲ），在ＨＶＲ内、外具有几个保守氨基酸残基和氨基酸区域，它们与外膜蛋白空间构型的维持有关。     

Genotype distribution and comparison of the putative envelope region of hepatitis C virus from Korean patients

Comparative nucleotide sequence studies of the genomes of hepatitis C virus (HCV) revealed that there are at least 6 different genotypes of HCV. The prevalence of HCV genotypes among the patients with liver diseases in Korea was investigated using the polymerase chain reaction (PCR) for the NS5 region. In the 75 HCV RNA positive samples, two genotypes, type 1b and type 2a, were the major causative agents which accounted for 60% and 33% of infections respectively, while 7% could not be assigned a genotype by the methods used. The nucleotide sequences of cDNAs encoding the putative envelope proteins from 10 type 1b and 5 type 2a genotype samples were analyzed. Approximately 31–42% of the nucleotide sequences of type 1b samples examined differed from those of different genotypes, In the case of type 2a samples, 36–42% of the nucleotide sequences differed from those of different genotypes. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. Two hypervariable regions (HVR1 and HVR2) were recognized in both HCV genomes of genotypes 1b and 2a. However, the sequence divergence within the HVR2 region of genotype 2a was less than that of genotype 1b. © 1995 Wiley-Liss, Inc.     

Absence of extensive genetic heterogeneity of hepatitis C virus in antibody-negative chronic hepatitis C

Hepatitis C virus (HCV) carriers usually have antibodies to HCV; however, there are viremic individuals without these antibodies. To investigate whether variations of the viral genome are responsible for this discrepancy, the nucleotide and deduced amino acid sequences of HCV capsid and nonstructural regions obtained from 15 viremic patients were examined. These 15 patients were infected with type 1b HCV, and 10 did not have antibody to HCV assayed with second-generation tests. The nucleotide homology of the 5 seropositive and 10 seronegative patients with the HCV prototype sequence were 91.6% and 91.9%, respectively, in the capsid region. There was no apparent difference in the deduced amino acid sequences between the two groups of patients studied (94% vs. 95%). The nucleotide and amino acid sequences of a part of the nonstructural region 3 also showed similar results. These findings suggest that absence of antibodies against both capsid and nonstructural peptides in HCV carriers is not caused by genetic heterogeneity of the viral epitopes. © 1996 Wiley-Liss, Inc.     

丙型肝炎病毒包膜区变异与感染慢性化关系的初步研究

目的 探讨丙型肝炎病毒(HCV)包膜区变异与其感染慢性化的关系。方法 3份HCV慢性感染者和3份急性感染者血标本，采用逆转录—聚合两链反应(RT—PCR)扩增HCV的E1区C端及E2区N端片段(573bp)，扩增产物进行克隆，以单链构象多态性(SSCP)和异质性双体(HD)分析对每份血清30个克隆的E1／E2区准种(quasispecies)进行筛选，挑选每份标本HCV的优势株与劣势株序列进行测定，分析非同义替换碱基数与同义替换碱基数比率(dN／dS，间接反映选择压力)和推导的氨基酸序列。结果 HCV慢性感染者病毒准种的复杂性和E2区dN／dS明显高于急性感染者。HCV慢性感染者的E2区氨基酸替换率(8．46％)比急性感染者(1．02％)更高，而两者的E1区氨基酸替换率(分别为2．74％和1．09％)均较低。尽管HVR1变异程度更高，但仍存在高度保守的氨基酸位点。结论 HCV持续性感染与准种复杂性增高和宿主对HVR1的免疫选择有关。     

Fluctuations of hepatitis C virus load are not related to amino acid substitutions in hypervariable region 1 and interferon sensitivity determining region.

Hepatitis C virus (HCV) load is one of the most important predictive factors of response to interferon treatment. However, little is known about the mode and determinants of viremia. The mode of viremia was investigated in 78 patients with chronic HCV genotype 1b infection during 1-2 years follow up. Virus load, determined by a branched chain DNA amplification assay, was stable in 73 of 78 (93.6%) patients, whereas 5 (6.4%) showed marked fluctuation (from undetectable level to more than 10 Meq/ml) in viral titer. To study the mechanisms mediating fluctuations in viral titer, amino acid sequences of two regions were examined; hypervariable region (HVR) 1 and the interferon sensitivity determining region (ISDR). Multiple amino acid substitutions were observed in HVR 1 but no relationship was evident between substitutions and virus titers. In contrast, no amino acid substitutions were observed in the ISDR in any patients with stable virus titer during a follow-up period of 12-24 months (7-24 samples) or in one patient who was observed for 15 years. Interestingly, multiple amino acid substitutions in the ISDR appeared in only two of the five patients with marked titer fluctuation, when the virus decreased markedly. Alanine aminotransferase levels in these five patients correlated with viral load. The data suggest that amino acid substitutions in HVR1 and ISDR are not essential for changes in viral titer. Possible mechanisms of fluctuations of viral titer and amino acid substitutions in the ISDR accompanying marked reductions in viral load are discussed.