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.     

Hepatitis C virus: The role of molecular mimicry in response to interferon treatment

Chronic hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In order for HCV to persist, the virus must escape immune recognition or inhibit the host immune response. The NS5A protein contains the interferon sensitivity‐determining region (ISDR) and is able to repress dsRNA‐dependent protein kinase (PKR) thus influencing the response to interferon (IFN) therapy. Patients who respond to IFN therapy have stronger antibody reactivity against the NS5A compared to IFN non‐responders. Therefore, given the possible role for the ISDR in IFN resistance and differential antibody reactivity, it is possible that variation in ISDR may be involved in viral immune escape and development of persistent HCV infection employing aspects of host mimicry. In this study, pre‐treatment samples obtained from HCV infected patients were used to investigate the effect of different NS5A ISDR variants on the IFN antiviral response and their involvement in immune evasion. The NS5A was identified as a homologue of the variable region of immunoglobulins (Ig). The IFN resistant genotypes had higher levels of similarity to Ig compared to IFN sensitive genotypes. Expression of NS5A‐6003 (HCV genotype 1b) and NS5A‐6074 (HCV genotype 2a) was able to rescue vesicular stomatitis virus (VSV) from IFN inhibition and restore luciferase activity. A correlation between Ig‐like NS5A structure and also antibody response with the outcome of IFN treatment was observed. J. Med. Virol. 84:1571–1585, 2012. © 2012 Wiley Periodicals, Inc.     

Mimotopes of the hyper variable region 1 of the hepatitis C virus induce cross-reactive antibodies directed against discontinuous epitopes.

Hepatitis C virus (HCV) is a major cause worldwide of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and the development of an effective vaccine represents a high priority goal. The hyper variable region 1 (HVR1) of the second envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. To be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1, selected from a specialized phage library using HCV patients' sera. Some of the cross-reacting anti-mimotope antibodies elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.     

Evasion of host immune surveillance by hepatitis C virus: potential roles in viral persistence

Hepatitis C virus (HCV) is a major human pathogen that causes mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. In order for a noncytopathic virus such as HCV to persist, the virus must escape immune recognition or evade host immune surveillance. Immune escape via the hypervariable region of the E2 envelope protein has been postulated as one mechanism for HCV persistent infection. Such hypervariability within the E2 protein may be under selective pressure from protective B cell or T cell responses and be able to escape immune recognition by rapid mutation of antigenic site. In addition to antigenic variation, HCV may also suppress immune response, leading to dampening of cellular immunity. This is supported by recent studies in our laboratory demonstrating that the HCV core protein can suppress host immune responses to vaccinia virus by downregulating viral specific cytotoxic T lymphocyte (CTL) responses and cytokine production. An understanding of the mechanisms behind HCV persistence will provide a basis for the rational design of vaccines and novel therapeutic agents targeting human HCV infection.     

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.     

Immunoglobulin allotypes influence IgG antibody responses to hepatitis C virus envelope proteins E1 and E2

Immunoglobulin (Ig) GM and KM allotypes—genetic markers of γ and κ chains, respectively—are associated with the outcome of hepatitis C virus (HCV) infection, but the underlying mechanisms are not well understood. We hypothesized that GM and KM allotypes could contribute to the outcome of HCV infection by influencing the levels of IgG antibodies to the HCV glycoproteins E1E2. We serologically allotyped 100 African American individuals with persistent HCV infection for GM and KM markers and measured anti-E1E2 antibodies. Subjects with the GM 1,17 5,13 phenotype had significantly higher levels of anti-E1E2 antibodies than subjects who lacked this phenotype (p = 0.008). Likewise, subjects with the KM 1–carrying phenotypes had higher levels of anti-E1E2 antibodies than subjects who lacked these phenotypes (p = 0.041). Median titers were fourfold higher in persons expressing both GM 1,17 5,13 and KM 1–carrying phenotypes compared with those who lacked these phenotypes (p = 0.011). Interactive effects of these GM-KM phenotypes were previously found to be highly significantly associated with spontaneous HCV clearance. Results presented here show that Ig allotypes contribute to the interindividual differences in humoral immunity to the HCV epitopes, a finding that may provide a mechanistic explanation for their involvement in the outcome of HCV infection.     

High prevalence of hypervariable region 1-specific and -cross-reactive CD4(+) T cells in HCV-infected individuals responsive to IFN-alpha treatment

The hypervariable region 1 (HVR1) of the putative envelope 2 protein of the hepatitis C virus (HCV) is the most variable part of the whole HCV polyprotein. Anti-HVR1 antibodies have been shown to protect against HCV infection, indicating that this region contains an important neutralization determinant. Recently we and others have demonstrated that HVR1 is also a T cell determinant able to activate helper T cell responses during HCV infection. In order to investigate the role of the immune response against HVR1 during HCV infection we have evaluated the humoral and lymphoproliferative responses to a panel of HVR1 peptides in HCV-infected patients with different outcomes of the disease following interferon-alpha (IFN-alpha) treatment. We observed that the frequency of anti-HVR1 T cell responses was significantly higher in patients who recovered after IFN-alpha therapy than in those who did not, while no differences in the anti-HVR1 antibody reactivities were detected. In addition, by generating HVR1-specific T cell lines and clones we identified human leukocyte-associated antigens DR4 restricted T cell epitopes in the carboxy-terminus of HVR1 and we demonstrated that broadly cross-reactive HVR1 T cells are elicited by HVR1.     

Characterization of antibody response to hepatitis C virus protein E2 and significance of hypervariable region 1-specific antibodies in viral neutralization

Summary.  Antibodies directed against hypervariable region 1 (HVR1) within the viral glycoprotein E2 of hepatitis C virus (HCV) are postulated to neutralize virus. An in vitro infection/binding assay of human fibroblast cells was established in order to study neutralization of HCV. Occurrence of mutations in the nucleotide sequence of HVR1 as compared to the inoculum after infection of human fibroblasts suggested replication of HCV in these cells. The significance of HVR1-specific antibodies in sera of patients who were infected in a single-source outbreak by an HCV contaminated anti-D immunoglobulin (IgG) preparation was studied. Using immunoprecipitation and ELISA, HVR1-specific antibodies could be detected in most of the sera obtained early (? 1 year p.i.) and late (up to 14 years p.i.) in single patients. Further characterization of the HVR1-specific antibodies in patient sera by attachment studies of HCV to the human fibro-blasts suggested that HVR1-specific antibodies in sera obtained early p.i. can neutralize virus of the anti-D IgG preparation.     

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.     

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。     

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.     

Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees

Summary.  A chimpanzee was immunized with two recombinant envelope glycoproteins E1 and E2 of hepatitis C virus (HCV), strain HCV-N2, and the hypervariable region 1 (HVR1) peptides of a different isolate, HCV-#6, then received an intravenous inoculation of 10 chimpanzee infectious doses of HCV-#6. With high humoral immune response against E1 and E2 but a low response against HVR1, the vaccinee became infected with the HCV. However, after increasing the titer of anti-HVR1 against HCV-#6, the vaccinee showed protection. Neutralization of HCV-#6 with the antiserum from this protected vaccinee was achieved by inoculation of this mixture into another chimpanzee. These results suggest that vaccination with a peptide-vaccine of homologous HVR1 is effective in the chimpanzee. Received December 4, 1998 Accepted January 12, 1999     

Induction of cross-reactive humoral immune response by immunization with mimotopes of the hypervariable region 1 of the hepatitis C virus

Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, worldwide, and the development of an effective vaccine represents a high priority goal. The Hyper Variable Region 1 (HVR1) of the second Envelope protein (E2) of HCV contains a principal neutralizing determinant, but it is highly variable among different isolates and it is involved in the escape from host immune response. Thus, to be effective, a vaccine should elicit a cross-reacting humoral response against the majority of viral variants. We show that it is possible to achieve a broadly cross-reactive immune response in rabbits by immunization with mimotopes of the HVR1. selected from a specialized phage library using HCV patients' sera. At least some of the cross-reacting anti-mimotope antibodies, elicited in rabbits, recognize discontinuous epitopes in a manner similar to those induced by the virus in infected patients.     

Delineation of Regions Important for Heteromeric Association of Hepatitis C Virus E1 and E2

Hepatitis C virus (HCV) is the major causative agent of blood-borne non-A non-B hepatitis. The persistence of HCV infection is believed to reflect escape from the host immunosurveillance system by mutations in hypervariable region 1 (HVR1) of the envelope protein 2 (E2). Two envelope proteins of HCV, E1 and E2, have been reported to form a heteromeric complex but the exact organization of the viral envelope proteins remains uncertain. We examined the interaction of E1 and E2 by far-Western blotting using the bacterial recombinant proteins and also by pull-down assay using mammalian expressed proteins. The major E1-interacting site of E2 was mapped within the N-terminal part of E2 (NCD1) (aa 415 to 500 of the polyprotein). Both HVR1 and HVR2, located at the N-terminal part of E2, were dispensable for the interaction. Although several discontinuous regions within NCD1 seemed to contribute to the strong binding to E1, the highly conserved amino acid sequences flanking HVR2 had the most significant effect. The amino acid residues “WHY” from 489 to 491 of E2 played an especially crucial role since the constructs with the internal deletion or substitution of the residues showed severely impaired E1-binding. The N-terminal part of E1 is important for the E2-binding as determined by far-Western blotting using the mammalian- and bacterial-expressed E2 proteins as probes. The mammalian-expressed, glycosylated forms of the E1 and E2 proteins exhibited E1–E2 binding activities similar to those of the bacterial-expressed, nonglycosylated forms in pull-down assays, suggesting that glycosylation is not prerequisite for the heteromeric complex formation of E1 and E2.     

Influence of the dynamics of the hypervariable region 1 of hepatitis C virus (HCV) on the histological severity of HCV recurrence after liver transplantation

Recurrence of hepatitis C virus (HCV) infection after liver transplantation is almost universal and usually leads to chronic hepatitis with different degrees of severity. The pathogenic mechanisms underlying the variable outcome of HCV infection recurrence are not well defined, but recent data suggest that the dynamics of HCV quasispecies may be involved. HCV quasispecies evolution was traced by longitudinal single strand conformation polymorphism, direct sequencing, and cloning analyses of pre- and post-transplant HCV-1b isolates from patients with histologically severe (seven cases) or mild or moderate (nine cases) HCV infection recurrence. Differences between the two groups of patients that concerned the level of viremia or the degree of HCV quasispecies complexity and diversity were not observed at any of the three time points analyzed. However, emergence of nucleotide and amino acid changes during the 12 months follow-up was significantly more frequent in patients with mild or moderate than in those with severe HCV infection recurrence. The ratio of non-synonymous to synonymous nucleotide substitutions 12 months after transplantation was also greater in the former, suggesting that the HVR1 of HCV is under stronger selective pressure in these subjects. These findings suggest that the degree of amino acid diversification in the HVR1 of HCV, which probably reflects the strength of immune pressure on HCV, is inversely related to the histological severity of HCV infection recurrence.     

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): Influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 ± 2.3 vs. 4.7 ± 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 ± 1.7 vs. 5.4 ± 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis C. J. Med. Virol. 54:256–264, 1998. © 1998 Wiley-Liss, Inc.     

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.     

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.     

Factors predictive of response to interferon-alpha therapy in hepatitis C virus type 1b infection

Interferon-alpha (IFN-alpha) has been used to treat hepatitis C Virus (HCV)-induced infection but has been effective in only about half of all patients. It is suggested that the different responses to IFN-alpha treatment in HCV infection may be influenced by HCV genotypes, HCV RNA titer at the beginning of IFN-alpha therapy, and the sequences of the interferon sensitivity determining region (ISDR). However, there have also been reports showing that these have no relation to an IFN-alpha effect. In a previous study, it was found that the nucleotide sequence variation in the hypervariable region (HVR) 1 of the HCV could predict the effect of IFN-alpha. In the present investigation, an attempt was made to determine the predictive factors of IFN-alpha therapy. Twenty-six patients with HCV infection were treated with IFN-alpha. Among these, 13 patients recovered after 3 to 6 months of IFN-alpha treatment, although the other 13 patients showed no response after 6 months of treatment with IFN-alpha. In order to determine the predictive factors of IFN-alpha therapy, the ALT levels, HCV genotypes, HCV serum titer, and the quasispecies of HVR 1 were compared between responders and non-responders. It is suggested that the variation in the HVR 1 and HCV serum titer can be used to predict the effect of IFN-alpha.