Multigene Tracking of Hepatitis C Virus Quasispecies after Liver Transplantation: Correlation of Genetic Diversification in the Envelope Region with Asymptomatic or Mild Disease Patterns

To investigate the role of hepatitis C virus (HCV) quasispecies mutation in the pathogenesis of HCV infection, we analyzed changes in the genetic diversity of HCV genomes in 22 patients before and after liver transplantation by using heteroduplex mobility assay (HMA) technology. All patients were infected with HCV genotype 1 and developed high-titer posttransplant viremia. Each patient was classified according to the severity of posttransplant hepatitis, as assessed by standard biochemical and histological criteria. HCV quasispecies were characterized by HMA analysis of eight separate subgenomic regions of HCV, which collectively comprise 44% of the entire genome. The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%). In contrast, genes encoding the core, NS4, and NS5b proteins had the least amount of genetic divergence after liver transplantation (range, 0.3 to 1.2%). The E1/E2 region showed the greatest change in genetic diversity after liver transplantation, and the change in HMRs was 2.5- to 3.3-fold greater in patients with asymptomatic or moderate disease than in those with severe disease. The E1-5′ region of HCV quasispecies isolated from patients in the asymptomatic group had a significantly greater degree of diversification after liver transplantation than the same regions of HCV quasispecies isolated from patients in the severe disease group (P = 0.05). While changes in the genetic diversity of some nonstructural genes were also greater in asymptomatic patients or in patients with mild disease than in patients with severe disease, the results were not significant. Data from this cohort demonstrate that greater rates of HCV quasispecies diversification are associated with mild or moderate liver disease activity in this immunosuppressed population.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is known to be a major causative agent of chronic liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (27). Chronic hepatitis C is now recognized as the leading indication for orthotopic liver transplantation in the United States, with nearly 100% of HCV-infected liver transplant recipients developing recurrent viremia after transplantation (4, 17, 22, 32, 44).The HCV genome is a single-stranded, positive-sense RNA of about 9.5 kb, which exists as a viral quasispecies in infected humans (6, 25, 31, 41). HCV quasispecies are characterized by extensive genetic mutation in the hypervariable region 1 (HVR1) of the second envelope glycoprotein gene (E2) (25, 41). Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms (15, 16, 29, 42). The role of evolution of HCV quasispecies in the development of chronic hepatitis C is currently unknown.HCV-infected liver transplant recipients offer an opportunity to study the evolution of HCV quasispecies in a new host tissue and to assess the role of quasispecies diversification in the development of posttransplant hepatitis. In a previous study (23) of HCV-infected liver transplant recipients, we found that in the three patients who developed severe, recurrent hepatitis, quasispecies major variants present in pretransplant serum samples were efficiently propagated after liver transplantation and during acute and chronic posttransplant hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum samples, followed by the emergence of quasispecies minor variants. These data suggested that the evolution of HCV quasispecies after liver transplantation may be related to posttransplant disease severity.In order to extend our previous findings and to address the hypothesis that mutation of other HCV genes also correlates with severity of posttransplant hepatitis C, we analyzed the pre- and posttransplant HCV quasispecies in 22 HCV-infected liver transplant recipients. The 22 patients were selected based on two virological criteria: all patients were infected with HCV genotype 1 before and after liver transplantation, and all patients developed recurrent, high-titer HCV viremia within 30 days posttransplant. Additionally, at least five posttransplant liver biopsies were available in each case to evaluate the histopathologic course of posttransplant hepatitis C. Pre- and posttransplant HCV quasispecies were characterized by using the heteroduplex mobility assay (HMA) to analyze eight different regions of the viral genome.     

Evolution of the Hepatitis C Virus Second Envelope Protein Hypervariable Region in Chronically Infected Patients Receiving Alpha Interferon Therapy

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.     

Follow-Up Study of Hypervariable Region Sequences of the Hepatitis C Virus (HCV) Genome in an Infant with Delayed Anti-HCV Antibody Responses

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti-HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti-HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.     

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.     

Nonrandom Distribution of Hepatitis C Virus Quasispecies in Plasma and Peripheral Blood Mononuclear Cell Subsets

The existence of an extrahepatic reservoir of hepatitis C virus (HCV) is suggested by differences in quasispecies composition between the liver, peripheral blood mononuclear cells, and serum. We studied HCV RNA compartmentalization in the plasma of nine patients, in CD19(+), CD8(+), and CD4(+) positively selected cells, and also in the negatively selected cell fraction (NF). HCV RNA was detected in all plasma samples, in seven of nine CD19(+), three of eight CD8(+), and one of nine CD4(+) cell samples, and in seven of eight NF cells. Cloning and sequencing of HVR1 in two patients showed a sequence grouping: quasispecies from a given compartment (all studied compartments for one patient and CD8(+) and NF for the other) were statistically more genetically like each other than like quasispecies from any other compartment. The characteristics of amino acid and nucleotide substitutions suggested the same structural constraints on HVR1, even in very divergent strains from the cellular compartments, and homogeneous selection pressure on the different compartments. These findings demonstrate the compartmental distribution of HCV quasispecies within peripheral blood cell subsets and have important implications for the study of extrahepatic HCV replication and interaction with the immune system.     

丙型肝炎病毒高变区基因变异特点初探

对两例ＨＣＶＲＮＡ持续阳性者分三个时间点随访五年,测定了ＨＣＶ的高变区基因序列,每个时间点平均测定２８个克隆,总共测定了１６８个克隆。研究发现ＨＣＶ高变区基因变异有五个明显特点：（１）变异程度大,高变区至少有８９％的核苷酸位点都可能发生变异;（２）变异形式多样,除常见的替代突变外,缺失突变（缺失１、２或３个核苷酸）的克隆数占总克隆数的２２．５％;（３）优势克隆明显,即有若干个克隆高变区的核苷酸和氨基酸序列相同;（４）类似株现象严重;（５）变异幅度大。该研究说明ＨＣＶ高变区基因变异具有高度复杂多样性。     

丙型肝炎病毒准种在NS2区的变异状况初探

探讨HCV准种在NS2区的基因结构特征及变异状况。利用逆转录－巢式PCR从1份HCV慢性携带者的阳性血清及1份丙肝患者的血清中获得HCV NS2全长cDNA,将其克隆于T载体,各随机挑取5个阳性克隆进行序列测定,结果显示克隆到HCV NS2全长基因,所测克隆在核苷酸水平和氨基酸水平互不相同。该慢性携带者HCV NS2区序列以完整读码框架（ORF）为主,一个于HCV多聚蛋白第835位氨基酸的位置出现终止信号,而该丙型肝炎患者以NS2N端发现终止信号的序列为主,其中三个于第835位氨基酸的位置出现终止信号,一个于第887位氨基酸的位置出现终止信号,仅一个克隆的序列为完整ORF。对ORF完整的序列进行比较,发现丙型肝炎患者氨基酸变异主要集中于N端,蛋白二级结构模拟显示丙肝患者NS2与慢性携带者的优势二级结构类似,研究表明从我们选择的两种感染者的HCV NS2序列看,不同临床类型的HCV病人体内的HCV准种在NS2区存在差异,这种差异可能与病毒存在于机体的状态一定的一致性。     

Differential amplification of hypervariable region 1 of hepatitis C virus by partially mismatched primers

The quasispecies nature of hepatitis C virus (HCV) has been well documented over its whole genome and the most variable domain is located at the 5' end of the second envelope region, the so-called hypervariable region 1 (HVR1). HVR1 has therefore been extensively used as the target for characterizing HCV quasispecies profiles. In this study, we reported our finding that partially mismatched primers preferentially amplify different HVR1 sequences in a heterogeneous virus population. This finding suggests a possible mechanism of bias during the amplification of HVR1 sequences and may be responsible for some conflicting data regarding evolutionary or clinical implications of HCV quasispecies.     

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.     

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.     

Unexpected maintenance of hepatitis C viral diversity following liver transplantation

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis in up to 20% of individuals, often requiring liver transplantation. Although the new liver is known to be rapidly reinfected, the dynamics and source of the reinfecting virus(es) are unclear, resulting in some confusion concerning the relationship between clinical outcome and viral characteristics. To clarify the dynamics of liver reinfection, longitudinal serum viral samples from 10 transplant patients were studied. Part of the E1/E2 region was sequenced, and advanced phylogenetic analysis methods were used in a multiparameter analysis to determine the history and ancestry of reinfecting lineages. Our results demonstrated the complexity of HCV evolutionary dynamics after liver transplantation, in which a large diverse population of viruses is transmitted and maintained for months to years. As many as 30 independent lineages in a single patient were found to reinfect the new liver. Several later posttransplant lineages were more closely related to older pretransplant viruses than to viruses detected immediately after transplantation. Although our data are consistent with a number of interpretations, the persistence of high viral genetic variation over long periods of time requires an active mechanism. We discuss possible scenarios, including frequency-dependent selection or variation in selective pressure among viral subpopulations, i.e., the population structure. The latter hypothesis, if correct, could have relevance to the success of newer direct-acting antiviral therapies.     

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.     

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants.     

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.     

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.     

Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.     

Pretransplant Serum Hepatitis C Virus RNA Levels Predict Response to Antiviral Treatment after Living Donor Liver Transplantation

Background

Given the limited efficacy and high adverse event rate associated with treatment of recurrent hepatitis C after liver transplantation, an individualized treatment strategy should be considered. The aim of this study was to identify predictors of response to antiviral therapy for hepatitis C after living donor liver transplantation (LDLT) and to study the associated adverse events.

Methods

A retrospective chart review was performed on 125 hepatitis C virus (HCV)-positive LDLT recipients who received interferon plus ribavirin and/or peginterferon plus ribavirin therapy at Kyoto University between January 2001 and June 2011.

Results

Serum HCV RNA reached undetectable levels within 48 weeks in 77 (62%) of 125 patients, and these patients were defined as showing virological response (VR). Of 117 patients, 50 (43%) achieved sustained VR (SVR). Predictive factors associated with both VR and SVR by univariate analysis included low pretransplant serum HCV RNA levels, a non-1 HCV genotype, and low pretreatment serum HCV RNA levels. In addition, LDLT from ABO-mismatched donors was significantly associated with VR, and white cell and neutrophil counts before interferon therapy were associated with SVR. Multivariate analysis showed that 2 variables–pretransplant serum HCV RNA level less than 500 kIU/mL and a non-1 HCV genotype–remained in models of both VR and SVR and that an ABO mismatch was associated with VR. No variables with a significant effect on treatment withdrawal were found.

Conclusions

Virological response to antiviral therapy in patients with hepatitis C recurring after LDLT can be predicted prior to transplant, based on pretransplant serum HCV-RNA levels and HCV genotype. LDLT from ABO-mismatched donors may contribute to more efficacious interferon therapy.

Trial Registration

UMIN-CTR UMIN000003286