Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.     

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.Hepatitis C virus (HCV) infection is a major global health issue leading to persistent viral infection in the majority of those infected and is associated with progressive liver disease, cirrhosis, and hepatocellular carcinoma. Six major genotypes of HCV have been described that have evolved in geographically distinct regions and that share approximately. 80% nucleotide homology with one another. HCV viral genotypes have been further classified into subtypes (25). HCV subtype 3a infection is now the most common subtype in the United Kingdom (11), although it is globally distributed and frequently associated with intravenous drug use.The classification of HCV viral strains by genotype and subtype has proven informative not only in terms of the epidemic and evolutionary history of the virus but also in terms of clinical outcomes. In particular, the response rates to current gold standard therapy (9) and the prevalence of hepatic steatosis (20) are significantly higher for subtype 3a than for genotype 1 infections. The reasons for this are not understood but must relate to viral genetic and phenotypic differences between strains, or to differences in the ability of hosts to exert an effective immune response against particular viral sequences, or to a combination of both factors.To date, detailed assessment of the HCV genome has largely focused on HCV genotype 1. Indeed, only a few full-length HCV subtype 3a viral sequences are currently published and available within the major HCV databases (Los Alamos; http://hcv.lanl.gov/components/hcv-db/combined_search/searchi.html and euHCVdb; http://euhcvdb.ibcp.fr/euHCVdb/) (16).To characterize HCV subtype 3a in detail, we performed whole-genome analysis of a cohort of patients with persistent HCV subtype 3a infection. We subsequently focus on the highly variable regions observed in the envelope protein E2 in both acute and chronic infection, since it was apparent that these regions were not restricted to the well-documented hypervariable region 1 (HVR1) that is found at the 5′ end of E2 in all HCV genotypes.Viral genomic variability can be assessed at a number of different levels; first, intergenotypic variability may arise in genomic regions that are conserved within the same subtype but are distinct between subtypes. Second, there is intragenotypic variability, which may be defined as regions of viral variability within the same genotype or subtype. Finally, intrahost variability is where viral genomic variability occurs within the same viral subtype and also the same host when individual clonal sequences are assessed. Although intergenotypic variability may simply be a feature of the existence of geographically distinct HCV subtypes, intragenotypic and intrahost variability may reflect viral regions subject to specific selection pressures, with important functional implications.We observed two distinct regions of intrahost and intragenotypic hypervariability within genotype 3a envelope 2 (E2)—in addition to the previously described HVR1—that we have named HVR495 and HVR575. We show that these regions are subject to positive selection pressure, sometimes very early in acute infection. Although HVR575 has been previously recognized as a site of intergenotypic variation (18), the identification of this region as a hypervariable site within genotype 3a and as a site under early selection pressure leading to variability within the same host has not been previously described.     

Hypervariable region 1 differentially impacts viability of hepatitis C virus strains of genotypes 1 to 6 and impairs virus neutralization

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a(ΔHVR1), 5a(ΔHVR1), and 6a(ΔHVR1) Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a(ΔHVR1), 1b(ΔHVR1), 2b(ΔHVR1), 3a(ΔHVR1), and 4a(ΔHVR1) recombinants were severely attenuated. However, except for recombinant 4a(ΔHVR1), viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a(ΔHVR1), 1b(ΔHVR1), 2b(ΔHVR1), and 3a(ΔHVR1) recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at ～1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a(ΔHVR1) produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.     

Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients.

The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed.     

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.     

An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies

Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins. To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with na?ve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV.     

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.     

Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(kappa)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The V(H) of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the V(L) kappa sequences were highly homologous. The affinity (K(d)) of 2P24 and 15H4 (10(-9) or 10(-8) M with two immunizing peptides and 10(-8) M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.     

Within-host dynamics of the hepatitis C virus quasispecies population in HIV-1/HCV coinfected patients

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3 × 10(-3) sub/site/month in group A and B and 0.29 × 10(-3) sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space.     

Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection

Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 x 10(-3) mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.     

Evolutionary rate and genetic drift of hepatitis C virus are not correlated with the host immune response: studies of infected donor-recipient clusters

Six donor-recipient clusters of hepatitis C virus (HCV)-infected individuals were studied. For five clusters the period of infection of the donor could be estimated, and for all six clusters the time of infection of the recipients from the donor via blood transfusion was also precisely known. Detailed phylogenetic analyses were carried out to investigate the genomic evolution of the viral quasispecies within infected individuals in each cluster. The molecular clock analysis showed that HCV quasispecies within a patient are evolving at the same rate and that donors that have been infected for longer time tend to have a lower evolutionary rate. Phylogenetic analysis based on the split decomposition method revealed different evolutionary patterns in different donor-recipient clusters. Reactivity of antibody against the first hypervariable region (HVR1) of HCV in donor and recipient sera was evaluated and correlated to the calculated evolutionary rate. Results indicate that anti-HVR1 reactivity was related more to the overall level of humoral immune response of the host than to the HVR1 sequence itself, suggesting that the particular sequence of the HVR1 peptides is not the determinant of reactivity. Moreover, no correlation was found between the evolutionary rate or the heterogeneity of the viral quasispecies in the patients and the strength of the immune response to HVR1 epitopes. Rather, the results seem to imply that genetic drift is less dependent on immune pressure than on the rate of evolution and that the genetic drift of HCV is independent of the host immune pressure.     

Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus

Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.     

Viral sequence evolution in acute hepatitis C virus infection

CD8(+)-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+ -T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total non-envelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of non-envelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.     

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.     

Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous in its primary sequence and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to immune escape and persistent infection. A binding site for neutralizing antibodies (Ab) has also been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from naturally-occurring HVR1 sequences showed promiscuous recognition of HVR1 variants in most patients via binding to C-terminal amino acid residues with conserved physicochemical properties. Monoclonal antibodies generated by immunization of mice with peptides derived from natural HVR1 sequences were shown to recognize several HVR1 variants in line with evidence gathered from studies using human sera. In addition, selected mAbs were able to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded sequences, and were shown to specifically capture circulating and recombinant HCV particles, suggesting that HVR1 is expressed on intact virus particles and therefore potentially able to interact with cellular receptor(s). These findings suggest that it is possible to induce a broadly reactive clonal immune response to multiple HCV variants and that this mechanism could be used in principle to induce protective immunity for a large repertoire of HCV variants.     

Escape from a dominant HLA-B*15-restricted CD8+ T cell response against hepatitis C virus requires compensatory mutations outside the epitope

Antiviral CD8(+) T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8(+) T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8(+) epitopes in NS5B (LLRHHNMVY(2450-2458) and SQRQKKVTF(2466-2474)), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs.     

Influence of increased CD4 cell counts on the genetic variability of hepatitis C virus in patients co-infected with human immunodeficiency virus I.

In order to study the effect of increased CD4 cell counts on the biology of hepatitis C virus (HCV), we analyzed the genetic variability of HCV generated over 8 y in eight human immunodeficiency virus-1 (HIV-1) and HCV co-infected patients. This was a retrospective study in which HIV patients were selected who had profound immune impairment evident over four years and were co-infected with HCV genotype 1 and who then went on highly active antiretroviral therapy (HAART). These patients achieved different degrees of immune reconstitution, measured as increased CD4 cell counts during a 4- to 8-y period, following initiation of HAART. HCV genetic variability was determined by measuring the genetic diversity (Hamming distance, HD), and complexity (number of viral variants) in plasma samples collected at yearly intervals just before and after the initiation of HAART. The parameters were assessed by molecular cloning and sequencing of a 575-bp fragment including the HCV envelope 1 and envelope 2 genes (E1/E2), containing the hypervariable region 1 (HVR1). significantly increased HVR1 genetic diversity was observed in analyzed samples where the patients' CD4 cell counts were > or =100 compared with CD4 cell counts <100. A significant increase in genetic diversity in HVR1 was detected in co-infected patients whose CD4 cell counts increased from <100 to >400 over a period of more than 4 y of HAART therapy. This was in contrast to a minimal increase in HCV genetic diversity of HVR1 occurring in patients whose CD4 cell counts failed to rise much over 200 over 7 y of follow up. Insertion and deletion of HCV genomic fragments in the E1/E2 region was documented in one patient who developed fulminant hepatitis C.     

Antigenicity and B-epitope mapping of hepatitis C virus envelope protein E2

Immunogenicity for laboratory animals (rabbits and mice) of the whole hepatitis C virus envelope proteins and their conserved as well as hypervariable HVR1 sites has been investigated. Rabbit immune responses to HCV envelope proteins (both single E2 and E1E2 heterodimer) were shown to be much more efficient than murine immune responses. Rabbit immunization with E2 protein caused formation of antibodies to several highly conserved linear B-epitopes of this protein as well as to the N-terminal fragment of the hypervariable region HVR1. Epitopes in the CR2 region were determined for the first time. There was cross-reactivity between the N-terminal fragment of the protein E2 hypervariable region HVR1 and the octapeptide fragment of the protein E1 conserved region CR1, which shared four identical amino acid residues.     

Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry

The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.