glycoprotein-e2--hepatitis-c-virus and Hepatitis-C--Chronic

The molecular mechanisms of hepatitis C virus (HCV) persistence and pathogenesis are poorly understood. The design of an effective HCV vaccine is challenging despite a robust humoral immune response against closely related strains of HCV. This is primarily because of the huge genetic diversity of HCV and the molecular evolution of various virus escape mechanisms. These mechanisms are steered by the presence of a high mutational rate in HCV, structural plasticity of the immunodominant regions on the virion surface of diverse HCV genotypes, and constant amino acid substitutions on key structural components of HCV envelope glycoproteins. Here, we review the molecular basis of neutralizing antibody (nAb)-mediated immune response against diverse HCV variants, HCV-steered humoral immune evasion strategies and explore the essential structural elements to consider for designing a universal HCV vaccine. Structural perspectives on key escape pathways mediated by a point mutation within the epitope, allosteric modulation of the epitope by distant mutations and glycan shift on envelope glycoproteins will be highlighted (abstract graphic).

Topics: Antibodies, Neutralizing; Antigens, Viral; Epitopes; Genetic Variation; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Evasion; Immunity, Humoral; Immunodominant Epitopes; Mutation; Protein Conformation; Protein Domains; Viral Envelope Proteins

Chronic hepatitis C virus (HCV) infection is the cause of about 400,000 annual liver disease-related deaths. The global spread of this important human pathogen can potentially be prevented through the development of a vaccine, but this challenge has proven difficult, and much remains unknown about the multitude of mechanisms by which this heterogeneous RNA virus evades inactivation by neutralizing antibodies (NAbs). The N-terminal motif of envelope protein 2 (E2), termed hypervariable region 1 (HVR1), changes rapidly in immunoglobulin-competent patients due to antibody-driven antigenic drift. HVR1 contains NAb epitopes and is directly involved in protecting diverse antibody-specific epitopes on E1, E2, and E1/E2 through incompletely understood mechanisms. The ability of HVR1 to protect HCV from NAbs appears linked with modulation of HCV entry co-receptor interactions. Thus, removal of HVR1 increases interaction with CD81, while altering interaction with scavenger receptor class B, type I (SR-BI) in a complex fashion, and decreasing interaction with low-density lipoprotein receptor. Despite intensive efforts this modulation of receptor interactions by HVR1 remains incompletely understood. SR-BI has received the most attention and it appears that HVR1 is involved in a multimodal HCV/SR-BI interaction involving high-density-lipoprotein associated ApoCI, which may prime the virus for later entry events by exposing conserved NAb epitopes, like those in the CD81 binding site. To fully elucidate the multifunctional role of HVR1 in HCV entry and NAb evasion, improved E1/E2 models and comparative studies with other NAb evasion strategies are needed. Derived knowledge may be instrumental in the development of a prophylactic HCV vaccine.

Topics: Amino Acid Motifs; Animals; Antibodies, Neutralizing; Hepacivirus; Hepatitis C, Chronic; Host-Pathogen Interactions; Humans; Immune Evasion; Viral Envelope Proteins; Virus Internalization

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.

Topics: Animals; Antibodies, Neutralizing; B-Lymphocytes; CD8-Positive T-Lymphocytes; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Evasion; Immunity, Humoral; Liver; Liver Transplantation; Viral Envelope Proteins; Virus Internalization

HCV is a major cause of chronic liver disease worldwide. There is no vaccine available and the current antiviral therapies fail to cure approximately half of treated patients. Liver disease caused by HCV infection is the most common indication for orthotopic liver transplantation. Unfortunately, reinfection of the new liver is universal and often results in an aggressive form of the disease leading to graft loss and the need for retransplantation. Immunotherapies using antibodies that potently inhibit HCV infection have the potential to control or even prevent graft reinfection. The virion envelope glycoproteins E1 and E2, which are involved in HCV entry into host cells, are the targets of neutralizing antibodies. To date, a number of monoclonal antibodies targeting conserved regions of E2 have been described that display outstanding neutralizing capabilities against HCV infection in both in vitro and in vivo systems. This article will summarize the current literature on these neutralizing anti-E2 antibodies and discuss their potential immunotherapeutic efficacy.

Topics: Antibodies, Neutralizing; Antibodies, Viral; Conserved Sequence; Epitopes; Hepacivirus; Hepatitis C, Chronic; Humans; Immunologic Factors; Immunotherapy; Viral Envelope Proteins

At present, about 3% of the human population are infected with hepatitis C virus (HCV). The first, acute stage of the disease is usually asymptomatic. However, only 15-25% of the infected eliminate the virus, while the remaining patients develop chronic hepatitis C (CHC). After 10-30 years of CHC, cirrhosis occurs in 20-30% of patients; 5-10% of this group eventually suffer from hepatocellular carcinoma. Unfortunately, up till now no effective methods protecting against HCV or allowing for efficient CHC treatment have been elaborated. This is primarily because not much is known about the mechanism of CHC emergence and the factors affecting anti-HCV therapy. There are several lines of evidence that some specific features of the virus, especially its high genetic variability might be responsible for the maintenance of HCV infection. Moreover, a few mechanisms which affect host-virus interactions and can additionally support CHC development have recently been identified. Hybridization between the host-encoded, liver-specific microRNA (miR-122) and the 5'-untranslated region of HCV genome was shown to be required for effective viral RNA replication. It was also postulated that HCV proteins mimic some of the human ones; that is why the virus is not eliminated. Another hypothesis assumes that interactions between HCV E2 glycoprotein and CD81 receptor modulate various cellular pathways, thus supporting viral propagation. There is no doubt that a better understanding of the mechanisms described above is of great importance for designing new therapeutic strategies and anti-HCV drugs.

Topics: Antiviral Agents; Drug Discovery; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Host-Pathogen Interactions; Humans; Liver; MicroRNAs; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins; Virus Replication

The ability of hepatitis C virus (HCV) to infect leukocytes could favour HCV pathogenesis. Although viral infection of these immunocompetent cells is poorly (or not) productive, the impact on their immunomodulatory functions could be important. Viral envelope glycoproteins E1 and E2, because of their crucial role in the recognition of viral receptors on permissive cells, could contribute to viral leukocytic tropism and, as a consequence, to the pathophysiology of HCV chronic infection.

Topics: Genes, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Leukocytes; RNA, Viral; Sequence Analysis, RNA; Structure-Activity Relationship; Viral Envelope Proteins; Viral Tropism

The present manuscript represents an updated review on different aspects of immunology involved during hepatitis C virus infection in human beings. This includes a brief mention of HCV structure, presentation of viral components to host immune system, and ensuing immune response and immunopathogenesis occurring during HCV infection. The present article also highlights immunodiagnosis of HCV infection and the current status of immunotherapy available for HCV eradication. Its envelope protein, E2, is the primary mediator of virus attachment and cell entry. CD81 molecule on cell surface acts as a major receptor for viral entry into the host cells. Mature dendritic cells play an important role in presenting viral antigen, activate T-cells, and initiate anti-viral immune response. Relative T-cell populations and release of different cytokines from activated T-cells ultimately determine the clearance or persistence of HCV viremia through cellular and humoral immune responses. Natural killer (NK) cells constitute the first line of host defense against invading viruses by recruiting virus-specific T-cells and inducing antiviral immunity in liver. Diagnosis of acute or chronic hepatitis C virus (HCV) infection is established by serological assays for presence of antibodies against different sets of viral proteins during varied periods post infection. An effective immunotherapy and vaccine against HCV is still awaited.

Topics: Antibodies, Viral; Antigen Presentation; Antigenic Variation; Antigens, CD; Cytokines; Dendritic Cells; Hepacivirus; Hepatitis C, Chronic; Humans; Immunity; Immunotherapy; Killer Cells, Natural; T-Lymphocyte Subsets; Tetraspanin 28; Viral Envelope Proteins; Virus Attachment

Topics: Antiviral Agents; Drug Resistance; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Immunity, Cellular; Interferons; Mutation; Protein Biosynthesis; RNA Helicases; Serine Endopeptidases; T-Lymphocytes, Cytotoxic; Transcription, Genetic; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

Hepatitis C virus(HCV), discovered in 1989, is the major causative agent of chronic viral hepatitis. Most patients progress to liver cirrhosis and hepatocellular carcinoma. In the therapy of hepatitis C, only interferon has been used effectively as an anti-HCV reagent in Japan, but its effectiveness is limited to about 30% of cases. Using human hepatocyte cell line which could support efficient HCV replication, we previously found that lactoferrin inhibited HCV infection, and demonstrated that this inhibiting activity was due to the interaction of lactoferrin with HCV. Further analysis found that the carboxyl region of lactoferrin, which partially shows amino acid sequence homology to human CD81, specifically bound to the HCV E2 envelope protein, and identified a 33 amino acids as a critical binding domain of lactoferrin. On the other hand, it has been shown that bovine lactoferrin was effective in some patients with chronic hepatitis received an 8-week course of lactoferrin treatment. Further clinical trials showed that lactoferrin is a promising candidate for adjuvant therapy with interferon in patients with chronic hepatitis C.

Topics: Antigens, CD; Clinical Trials as Topic; Drug Therapy, Combination; Hepatitis C, Chronic; Humans; Interferon-alpha; Lactoferrin; Membrane Proteins; Protein Binding; Sequence Homology, Amino Acid; Tetraspanin 28; Viral Envelope Proteins

Chronic hepatitis C virus (HCV) infection is the most common cause of end-stage liver disease, often leading to liver transplantation, in which case circulating virions typically infect the transplanted liver within hours and viral concentrations can quickly exceed pre-transplant levels. MBL-HCV1 is a fully human monoclonal antibody recognizing a linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423). The ability of MBL-HCV1 to prevent HCV recurrence after liver transplantation was investigated in a phase 2 randomized clinical trial evaluating six MBL-HCV1-treated subjects and five placebo-treated subjects. MBL-HCV1 treatment significantly delayed time to viral rebound compared with placebo treatment. Here we report results from high-throughput sequencing on the serum of each of the eleven enrolled subjects prior to liver transplantation and after viral rebound. We further sequenced the sera of the MBL-HCV1-treated subjects at various interim time points to study the evolution of antibody-resistant viral variants. We detected mutations at one of two positions within the antibody epitope--mutations of N at position 415 to D, K or S, or mutation of N at position 417 to S. It has been previously reported that N415 is not glycosylated in the wild-type E2 protein, but N417S can lead to glycosylation at position 415. Thus N415 is a key position for antibody recognition and the only routes we identified for viral escape, within the constraints of HCV fitness in vivo, involve mutating or glycosylating this position. Evaluation of mutations along the entire E1 and E2 proteins revealed additional positions that changed moderately before and after MBL-HCV1 treatment for subsets of the six subjects, yet underscored the relative importance of position 415 in MBL-HCV1 resistance.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Biological Evolution; Biomarkers; Double-Blind Method; Follow-Up Studies; Glycosylation; Hepatitis C, Chronic; High-Throughput Nucleotide Sequencing; Humans; Liver Transplantation; Molecular Sequence Data; Prognosis; Recurrence; RNA, Viral; Sequence Homology, Amino Acid; Viral Envelope Proteins

The adaptive immune response against hepatitis C virus (HCV) is significantly shaped by the host's composition of HLA-alleles with the consequence that the HLA phenotype is a critical determinant of viral evolution during adaptive immune pressure. In the present study, we aimed to identify associations of HLA class I alleles with HCV subtypes 1a and 1b genetic variants.. The association between HCV genetic variants and specific HLA-alleles was investigated in a cohort of 159 patients with chronic HCV genotypes 1a- and 1b-infection who were treated with pegylated interferon-alfa 2b and ribavirin in a prospective controlled trial for 48 weeks by direct sequencing of the genes encoding the HCV proteins E2, NS3, and NS5B and by HLA class I-genotyping of patients. HCV genetic variants were associated with specific HLA-alleles and the binding strength of accordant amino acid sequences to the corresponding HLA-allele was assessed by using the SYFPEITHI-algorithm.. Overall, associations between HLA class I alleles and HCV sequence variation were rare. Five unknown HLA class I-associated viral genetic variations were identified, which in part affected the binding of predicted HCV CD8+ T cell epitopes to the respective HLA-allele. In addition, different patterns of HLA class I-allele/HCV sequence associations between the two subtypes were observed.. We identified several unknown HLA class I-restricted HCV variants which in part impair binding to predicted HCV CD8+ T cell epitopes with remarkable differences between HCV subtypes 1a and 1b quasispecies.

Topics: Adolescent; Adult; Aged; Alleles; Base Sequence; CD8-Positive T-Lymphocytes; Cohort Studies; DNA Primers; Female; Genes, MHC Class I; Genetic Association Studies; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Host-Pathogen Interactions; Humans; Male; Middle Aged; Phylogeny; Prospective Studies; RNA, Viral; Viral Envelope Proteins; Viral Nonstructural Proteins; Young Adult

The hepatitis C virus (HCV) envelope 2 (E2) protein interacts with the cellular receptor CD81 in vitro. Within E2, 2 CD81-binding sites were described. E2-CD81 interaction has been shown to modulate B and T cell function. The clinical importance of mutations within the CD81-binding sites and overlapping hypervariable region 2 (HVR2) in correlation with response to antiviral treatment is unknown. Fifty-five patients infected with HCV-1b or HCV-3a underwent interferon-alpha-based treatment. The E2 gene, comprising the CD81-binding sites and HVR2, was sequenced from pretreatment serum samples. The number of mutations within CD81-binding sites was not correlated with virologic treatment response in HCV-1b- and HCV-3a-infected patients. Within HVR2, the total number of mutations was significantly higher in HCV-1b-infected patients with a sustained response to interferon-alpha-based treatment (3.9; range, 1-6) than in those with relapse (2.9; range, 1-5) or those who did not respond (2.8; range, 1-5) (P = .041). However, when the same analyses were based only on functionally nonconserved mutations, no significant differences were observed.

Topics: Adult; Amino Acid Sequence; Antigens, CD; Antiviral Agents; Binding Sites; Cloning, Molecular; Complementarity Determining Regions; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Membrane Proteins; Middle Aged; Molecular Sequence Data; Mutation; Receptors, Virus; Sequence Alignment; Tetraspanin 28; Treatment Outcome; Viral Envelope Proteins

The heterogeneity of the envelope 2 (E2) gene of the hepatitis C virus (HCV) was involved in the sensitivity of HCV to interferon-alpha (IFN-alpha). To assess the factors leading to virus eradication by IFN-alpha, patients whose first treatment by IFN-alpha failed and who had virus eradication after a second treatment were studied. These patients were paired with subjects in whom both treatments failed. The phosphorylation homology domain of the E2 gene (E2-PHD) had no sequence variation between the two stages in both groups of patients. Therefore, this region has no clinical predictive value within a specific genotype. The hypervariable region 1 (HVR1) was analyzed by cloning and sequencing 20 clones per sample. Comparison of samples showed that the change in quasispecies induced by the first IFN-alpha therapy could be associated with virus elimination obtained after a second treatment. The greater proportion of nonsynonymous mutations that was noted before the second treatment in responders suggest that pretherapeutic immune response is a major factor determining virus elimination and that the immune status of these patients changed between the first and the second treatment.

Topics: Adult; Aged; Amino Acid Sequence; Base Sequence; DNA, Viral; Female; Genes, env; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon Type I; Male; Middle Aged; Molecular Sequence Data; Mutation; Recombinant Proteins; Retrospective Studies; Sequence Homology, Amino Acid; Treatment Failure; Viral Envelope Proteins; Viral Proteins

Interferon (IFN) alfa has been used widely for the treatment of chronic hepatitis C virus (HCV) infections but only a small number of patients treated have shown a sustained biochemical and virological response. Anti-envelope E1 and E2 antibody titers were assessed retrospectively before, during, and after treatment with IFN in order to evaluate their usefulness for the prediction and monitoring of therapy outcome in 115 patients infected chronically with HCV genotype 1b. At baseline, E2 induced more frequent and stronger immunogenic responses than E1, irrespective of patient response to therapy. E1 and E2 antibodies also tended to be higher in patients with a long-term or a transient response to IFN treatment than in patients who were absolute non-responders. In most patients, E1 and E2 antibody levels tended to be lower after treatment. This reduction was most pronounced and occurred most frequently in long-term responders to therapy. In this patient group, the reduction of E1 antibodies was more pronounced than that of E2 antibodies. In contrast to E2 antibodies, the decrease of E1 antibodies could already be observed at the end of therapy (week 24) and was significantly larger (p<0.05) than that observed in relapsers and non-responders. Thus, a sustained elevation of E1 antibodies seems to be associated with ongoing infection even when HCV RNA levels were undetectable in serum. Monitoring of E1 antibody titers may represent a useful additional marker to discriminate sustained responders from those who relapse in patients receiving interferon therapy.

Topics: Antigens, Viral; Antiviral Agents; Genotype; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Interferon alpha-2; Interferon-alpha; Monitoring, Immunologic; Recombinant Proteins; Retrospective Studies; RNA, Viral; Viral Envelope Proteins

The extremely high rate of chronicity to hepatitis C virus (HVC) infection suggests an inefficient immune response. The humoral immune response to HCV was evaluated in 60 patients with chronic HCV infection and in 12 patients acutely infected with HCV.. A number of recombinant HCV antigens including the core, envelope 2 (E2), nonstructural (NS) 3, NS4, and NS5 proteins, and NS4a and E2-HVR-1 peptides were used in enzyme-linked immunoassays.. Immunoglobulin (Ig) G antibody responses to these viral antigens, except for the HCV core, were highly restricted to the IgG1 isotype. The prevalence of antibodies of the IgG1 isotype specific for the HCV core, E2, E2-HVR1, NS3 (helicase domain), NS4, and NS5 antigens was 97%, 98%, 28%, 88%, 33%, and 68%, respectively. Antibodies of the IgG3 isotype specific for E2, E2-HVR-1, NS3, NS4, and NS5 were detected in a minority of serum samples. The IgG2 and IgG4 isotypes were rarely if ever detected. Furthermore, antibody responses to HCV viral antigens were of relatively low titer and, with the exception of anti-HCV core, were delayed in appearance until the chronic phase of infection.. The IgG1 restriction, low titer, and delayed appearance of antibody responses elicited during HCV infection suggest that the immunogenicity of HCV proteins is limited in the context of natural infection. Inasmuch as recombinant HCV viral antigens perform as relatively normal immunogens in small animals, we suggest that the defective humoral immune responses during HCV infection may be attributable to an "immune avoidance" strategy.

Topics: Acute Disease; Antibody Formation; Enzyme-Linked Immunosorbent Assay; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Immunoglobulin G; Kinetics; Viral Core Proteins; Viral Envelope Proteins

Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker.. The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights.. AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed.. These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Topics: Animals; Antiviral Agents; Disease Models, Animal; Female; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; Immunoconjugates; Lectins; Liver; Male; Mice; Polysaccharides; Recombinant Fusion Proteins; Transplantation Chimera; Viral Envelope Proteins

Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on

Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Epitopes; Evolution, Molecular; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Evasion; Neutralization Tests; Quasispecies; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins; Viral Structural Proteins

Hepatitis C virus (HCV) is one of very few viruses that are either naturally cleared, or alternatively persist to cause chronic disease. Viral diversity and escape, as well as host adaptive immune factors, are believed to control the outcome. To date, there is limited understanding of the critical, early host-pathogen interactions. The asymptomatic nature of early HCV infection generally prevents identification of the transmitted/founder (T/F) virus, and thus the study of host responses directed against the autologous T/F strain. In this study, 14 rare subjects identified from very early in infection (4-45 days) with varied disease outcomes (n = 7 clearers) were examined in regard to the timing, breadth, and magnitude of the neutralizing antibody (nAb) response, as well as evolution of the T/F strain. Clearance was associated with earlier onset and more potent nAb responses appearing at a mean of 71 days post-infection (DPI), but these responses were narrowly directed against the autologous T/F virus or closely related variants. In contrast, a delayed onset of nAbs (mean 425 DPI) was observed in chronic progressors that appear to have targeted longitudinal variants rather than the T/F strain. The nAb responses in the chronic progressors mapped to known CD81 binding epitopes, and were associated with rapid emergence of new viral variants with reduced CD81 binding. We propose that the prolonged period of viremia in the absence of nAbs in these subjects was associated with an increase in viral diversity, affording the virus greater options to escape nAb pressure once it emerged. These findings indicate that timing of the nAb response is essential for clearance. Further investigation of the specificities of the early nAbs and the factors regulating early induction of protective nAbs is needed.

Topics: Adult; Antibodies, Neutralizing; Antibody Formation; Epitopes; Female; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Host-Pathogen Interactions; Humans; Longitudinal Studies; Male; Tetraspanin 28; Viral Envelope Proteins; Viremia; Young Adult

Hepatitis C virus (HCV) infection remains a major public health issue despite the introduction of several direct‑acting antiviral agents (DAAs), with some 185 million individuals infected with HCV worldwide. There is an urgent need for an effective prophylactic HCV vaccine. In the present study, we constructed genetic vaccines based on novel recombinant adeno‑associated viral (rAAV) vectors (AAV2/8 or AAV2/rh32.33) that express the envelope glycoprotein E2 from the HCV genotype 1b. Expression of HCV E2 protein in 293 cells was confirmed by western blot analysis. rAAV2/8.HCV E2 vaccine or rAAV2/rh32.33.HCV E2 vaccine was intramuscularly injected into C57BL/6 mice. HCV E2‑specific antigen was produced, and long‑lasting specific antibody responses remained detectable XVI weeks following immunization. In addition, the rAAV2/rh32.33 vaccine induced higher antigen‑specific antibody levels than the rAAV2/8 vaccine or AAV plasmid. Moreover, both AAV vaccines induced neutralizing antibodies against HCV genotypes 1a and 1b. Finally, it is worth mentioning that neutralizing antibody levels directed against AAV2/rh32.33 were lower than those against AAV2/8 in both mouse and human serum. These results demonstrate that AAV vectors, especially the AAVrh32.33, have particularly favorable immunogenicity for development into an effective HCV vaccine.

Topics: Adult; Animals; Antibodies, Neutralizing; Dependovirus; Female; Genetic Vectors; HEK293 Cells; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Sera; Immunity, Humoral; Immunization; Male; Mice; Mice, Inbred C57BL; Middle Aged; Recombinant Proteins; Vaccines, DNA; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) is the major causative agent of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The recent development of highly effective direct-acting antivirals (DAAs) has revolutionized the treatment of HCV patients. However, these DAAs are exorbitantly expensive for the majority of HCV patients worldwide. Moreover, these drugs still show genotypic difference in cure rate and have some resistant-associated variants. Tylophorine, a natural compound derived from Tylophora indica plants, is known to have anti-inflammatory and anti-cancerous growth activities. In the present study, we showed that two tylophorine intermediates, 5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl) methyl]-L-proline (O859585) and 2,3,6,7-tetramethoxy-9-phenanthrenecarboxylic acid (T298875), displayed anti-HCV activity with an EC

Topics: Alkaloids; Antiviral Agents; Cell Line, Tumor; Cell Survival; Drug Synergism; Drug Therapy, Combination; HEK293 Cells; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; Indolizines; Interferon-alpha; Phenanthrenes; Primary Cell Culture; Proline; Sofosbuvir; Tetraspanin 28; Tylophora; Viral Envelope Proteins; Virus Internalization

Neutralizing antibodies can prevent hepatitis C virus (HCV) infection, one of the leading causes of cirrhosis and liver cancer. Here, we characterized the immunoglobulin repertoire of memory B-cell antibodies against a linear epitope in the central front layer of the HCV envelope (E2; amino acids 483-499) in patients who were infected in a single-source outbreak. A reverse transcription polymerase chain reaction-based immunoglobulin gene cloning and recombinant expression approach was used to express monoclonal antibodies from HCV E2 peptide-binding immunoglobulin G-positive memory B cells. We identified highly mutated antibodies with a neutralizing effect in vitro against different genotype isolates sharing similar gene features. Our data confirm the importance of VH1-69 use for neutralizing activity. The data offer a promising basis for vaccine research and the use of anti-E2 antibodies as a means of passive immunization.

Topics: Adult; Aged; Antibodies, Monoclonal; B-Lymphocytes; Broadly Neutralizing Antibodies; Cohort Studies; Epitopes, B-Lymphocyte; Female; Genotype; HEK293 Cells; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoglobulin G; Immunologic Memory; Male; Middle Aged; Rho(D) Immune Globulin; Single-Domain Antibodies; Viral Envelope Proteins; Viral Hepatitis Vaccines

Hepatitis C virus (HCV) infection is a major global public health problem. Early induction of cross-reactive neutralizing antibodies during acute infection correlates with the spontaneous clearance of HCV. Understanding the antibody response in multiple subjects in large-scale studies would greatly benefit vaccine development. To determine the breadth of a polyclonal-serum antibody response, and or, the monoclonal antibodies against the different HCV E1E2 genotypes, we developed a quick and high throughput flow cytometry assay using fluorescent cell barcoding to distinguish cells transfected with different E1E2 sequences in a single measurement. HCV-specific antibodies recognizing conformational epitopes were tested for binding to cells transfected with E1E2 from six genotypes. In this assay, 1500 samples can be analyzed for specific binding to 6 different HCV E1E2 sequences within 8h. Plasma of HCV infected subjects were tested in our assay allowing us to determine the breadth of their antibody response. In summary, we developed a quick and high throughput assay to study the specificity of an antibody response against multiple HCV E1E2 sequences simultaneously. This assay can also be used to facilitate the discovery of novel antibodies, and because other flavi- and picornaviruses have similar intracellular assembly mechanisms, this approach can be used to study the antibody response against such viruses.

Topics: Antibodies, Neutralizing; Antibody Formation; Cell Separation; Cross Reactions; Epitopes, B-Lymphocyte; Flow Cytometry; Fluorescence; HEK293 Cells; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; High-Throughput Screening Assays; Humans; Neutralization Tests; Transgenes; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major public health concern, with over 70 million people infected worldwide, who are at risk for developing life-threatening liver disease. No vaccine is available, and immunity against the virus is not well-understood. Following the acute stage, HCV usually causes chronic infections. However, ~30% of infected individuals spontaneously clear the virus. Therefore, using HCV as a model for comparing immune responses between spontaneous clearer (SC) and chronically infected (CI) individuals may empower the identification of mechanisms governing viral infection outcomes. Here, we provide the first in-depth analysis of adaptive immune receptor repertoires in individuals with current or past HCV infection. We demonstrate that SC individuals, in contrast to CI patients, develop clusters of antibodies with distinct properties. These antibodies' characteristics were used in a machine learning framework to accurately predict infection outcome. Using combinatorial antibody phage display library technology, we identified HCV-specific antibody sequences. By integrating these data with the repertoire analysis, we constructed two antibodies characterized by high neutralization breadth, which are associated with clearance. This study provides insight into the nature of effective immune response against HCV and demonstrates an innovative approach for constructing antibodies correlating with successful infection clearance. It may have clinical implications for prognosis of the future status of infection, and the design of effective immunotherapies and a vaccine for HCV.

Topics: Antibodies, Neutralizing; Cell Line, Tumor; Computational Biology; Datasets as Topic; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; High-Throughput Nucleotide Sequencing; Humans; Machine Learning; Peptide Library; Prognosis; Remission, Spontaneous; Viral Envelope Proteins

The humoral immune system responds to chronic hepatitis C virus (HCV) infection by producing neutralising antibodies (nAb). In this study we generated three HCV pseudoparticles in which E1E2 glycoprotein sequence was targeted by the host humoral immune system. We used patient derived virus free Fabs (VF-Fabs) obtained from HCV genotype 1a (n = 3), genotype 1b (n = 7) and genotype 3a (n = 1) for neutralisation of HCVpp produced in this study both individually and in combination. Based on the available anti-HCV monoclonal nAb mapping information we selected amino acid region 384-619 for conformational epitope mapping. Amongst our notable findings, we observed significant reduction in HCVpp infectivity (p<0.05) when challenged with a combination of inter genotype and subtype VF-Fabs. We also identified five binding motifs targeted by patient derived VF-Fab upon peptide mapping, of which two shared the residues with previously reported epitopes. One epitope lies within an immunodominant HVR1 and two were novel. In summary, we used a reverse epitope mapping strategy to identify preferred epitopes by the host humoral immune system. Additionally, we have combined different VF-Fabs to further reduce the HCVpp infectivity. Our data indicates that combining the antigen specificity of antibodies may be a useful strategy to reduce (in-vitro) infectivity.

Topics: Antibodies, Viral; Epitope Mapping; Hepacivirus; Hepatitis C, Chronic; Humans; Neutralization Tests; Viral Envelope Proteins; Virulence

Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (<180days) and chronic (>180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

Topics: Acute Disease; Amino Acid Sequence; Antibodies, Monoclonal; Antibodies, Neutralizing; Epitope Mapping; Epitopes; Gene Expression; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immune Evasion; Models, Molecular; Mutation Rate; Protein Structure, Secondary; Viral Envelope Proteins

Hypervariable region 1 (HVR1) is one of the potential neutralization domains in the E2 glycoprotein of hepatitis C virus (HCV). Point mutations of the HVR1 can lead to humoral immune escape in HCV-infected patients. In this study, we segregated the chronically infected viraemic sera from HCV-infected patients into populations of antibody-free virus and antibody-associated virus (AAV) and mapped potential epitopes within the E1E2 gene junction of AAV sequences (residues 364-430). Furthermore, we generated HCV pseudoparticles (HCVpp) derived from AAV sequences to assess their infectivity. We studied the neutralization potential of virus-free Fab obtained from antibody-virus complexes, in the HCVpp system. We observed selective targeting of clonotypic HCV variants from the quasispecies pool. Moreover, we identified potential neutralizing epitopes within the HVR1 and an additional epitope that overlapped with a broadly neutralizing AP33 epitope (amino acid 412-423 in E2). We observed a marked difference in the infectivity of HCVpp generated using E1E2 sequences isolated from AAV. We document reduction in the infectivity of HCVpp-H77 and HCVpp derived from AAV sequences when challenged with virus-free Fab. Our results provide novel insights into the complexities of engagement between HCV and the humoral immune system.

Topics: Amino Acid Sequence; Endopeptidase K; Epitope Mapping; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunity, Humoral; Immunoglobulin Fab Fragments; Neutralization Tests; Serum; Viral Envelope Proteins; Viral Proteins; Viremia

Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders.. Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.

Topics: B-Lymphocytes; Cell Line; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; Transduction, Genetic; Viral Envelope Proteins; Viral Tropism; Virus Internalization

Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3'-end of E1 highly conserved and the 5'-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone.

Topics: Epistasis, Genetic; Evolution, Molecular; Hepacivirus; Hepatitis C, Chronic; High-Throughput Nucleotide Sequencing; Humans; RNA, Viral; Viral Envelope Proteins; Virion

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) comprises the first 27 N-terminal amino acid residues of E2. It is classically seen as the most heterogeneous region of the HCV genome. In this study, we assessed HVR1 evolution by using ultradeep pyrosequencing for a cohort of treatment-naive, chronically infected patients over a short, 16-week period. Organization of the sequence set into connected components that represented single nucleotide substitution events revealed a network dominated by highly connected, centrally positioned master sequences. HVR1 phenotypes were observed to be under strong purifying (stationary) and strong positive (antigenic drift) selection pressures, which were coincident with advancing patient age and cirrhosis of the liver. It followed that stationary viromes were dominated by a single HVR1 variant surrounded by minor variants comprised from conservative single amino acid substitution events. We present evidence to suggest that neutralization antibody efficacy was diminished for stationary-virome HVR1 variants. Our results identify the HVR1 network structure during chronic infection as the preferential dominance of a single variant within a narrow sequence space.. HCV infection is often asymptomatic, and chronic infection is generally well established in advance of initial diagnosis and subsequent treatment. HVR1 can undergo rapid sequence evolution during acute infection, and the variant pool is typically seen to diverge away from ancestral sequences as infection progresses from the acute to the chronic phase. In this report, we describe HVR1 viromes in chronically infected patients that are defined by a dominant epitope located centrally within a narrow variant pool. Our findings suggest that weakened humoral immune activity, as a consequence of persistent chronic infection, allows for the acquisition and maintenance of host-specific adaptive mutations at HVR1 that reflect virus fitness.

Topics: Adult; Aged; Aging; Amino Acid Sequence; Amino Acid Substitution; Antibodies, Neutralizing; Base Sequence; Female; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunity, Humoral; Immunoglobulin G; Liver Cirrhosis; Male; Middle Aged; Molecular Sequence Data; Sequence Analysis, RNA; Viral Envelope Proteins; Viral Proteins; Young Adult

Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment.. HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann-Whitney and Fisher's exact tests.. Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%.. While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters.

Topics: Adult; Antiviral Agents; Base Sequence; Female; Genetic Heterogeneity; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; High-Throughput Nucleotide Sequencing; Humans; Interferon-alpha; Male; Middle Aged; Prospective Studies; Recombinant Proteins; Ribavirin; Treatment Outcome; Viral Envelope Proteins

The correlation of neutralizing antibodies to treatment outcome in patients with chronic hepatitis C virus (HCV) infection has not been established. The aim of this study was to determine whether neutralizing antibodies could be used as an outcome predictor in patients with chronic HCV, genotype 1, infection treated with pegylated interferon-α and ribavirin. Thirty-nine patients with chronic hepatitis C, genotype 1a or 1b, with either sustained virologic response (n = 23) or non-sustained virologic response (n = 16) were enrolled. Samples taken prior to treatment were tested for their ability to neutralize 6 different HCV genotype 1 cell culture recombinants (1a: H77/JFH1, TN/JFH1, DH6/JFH1; 1b: J4/JFH1, DH1/JFH1, DH5/JFH1). The results were expressed as the highest dilution yielding 50% neutralization (NAb50-titer). We observed no genotype or subtype specific differences in NAb50-titers between patients with chronic HCV infection with and without sustained virologic response when tested against any of the included culture viruses. However, NAb50-titers varied significantly with a mean reciprocal NAb50-titer of 800 (range: 100-6400) against DH6/JFH1 compared to a mean NAb50-titer of 50 (range: <50-400) against all other included isolates. Subsequent studies demonstrated that the efficient neutralization of DH6/JFH1 could be linked to engineered adaptive mutations in the envelope-2 protein. In analysis of envelope 1 and 2 sequences of HCV, recovered from a subset of patients, we observed no apparent link between relatedness of patient sequences with culture viruses used and the corresponding neutralization results. In conclusion, pre-treatment levels of neutralizing antibodies against HCV genotype 1 isolates could not predict treatment outcome in patients with chronic HCV infection. High neutralization susceptibility of DH6/JFH1 could be correlated with adaptive envelope mutations previously highlighted as important for neutralization. Our study emphasizes the importance of using multiple culture viruses for neutralization studies and contributes to the current knowledge about neutralizing epitopes, important for future therapeutic- and vaccine-studies.

Topics: Antibodies, Neutralizing; Culture Techniques; DNA, Recombinant; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Mutation; Ribavirin; Treatment Outcome; Viral Envelope Proteins

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regions encompassing amino acids 313-327 and 432-443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of which were from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes. The remaining 126 samples were isolated from patients who had spontaneously cleared HCV infection. In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313-327 and 432-443 was 24.29% (51 of 210) and 4.76% (10 of 210), respectively. In the spontaneous clearance group (SC group), the prevalence was 0.79% (1 of 126) and 12.70% (16 of 126), respectively. The positive serum samples that contained antibodies reactive to epitope 313-327 neutralized HCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313-327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432-443 had cross-genotype neutralizing activities. The neutralizing activity of SC38, SC86, SC92 and CHC75 was partially inhibited by peptide 432-443. However, the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVpp were not inhibited by the peptide. This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope 432-443 as sources for future antibody therapies.

Topics: Amino Acid Sequence; Amino Acids; Antibodies, Neutralizing; Antibodies, Viral; Antibody Formation; Antibody Specificity; Cells, Cultured; Demography; Epitopes; Female; Genotype; Hepatitis C; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Neutralization Tests; Peptides; Sequence Alignment; Viral Envelope Proteins

Half of the patients chronically infected with hepatitis C virus (HCV) genotype 1 fail to respond to pegylated interferon alpha (PEG-IFN) and ribavirin (RBV) therapy. This study assesses the effects of treatment on the evolution of the E1-E2 viral region in non-responder patients infected with HCV-1b. Twenty-three HCV-1b chronically infected patients were studied retrospectively, including 19 non-responders to PEG-IFN/RBV therapy (11 null-responders and 8 relapsers) in the study group, and 4 untreated patients in the control group. Genetic and phylogenetic analyses of the E1-E2 viral populations were performed at baseline and at the time of treatment failure to assess changes in genetic variability and evolutionary dynamics during treatment. Baseline virological characteristics were similar in null-responders, relapsers and controls. E1-E2 genetic variability decreased during treatment in non-responders, with a more pronounced decline in relapsers than in null-responders. A specific evolutionary pattern was not observed in null-responders, while a complete substitution of viral variants found at baseline characterised relapser patients. No specific E1-E2 amino acid substitution involved in treatment failure could be identified. In conclusion, although diverse evolutionary patterns with no apparent common adaptive changes were observed during therapy, treatment failure was characterised by a decline in genetic diversity.

Topics: Adaptation, Biological; Adult; Aged; Amino Acid Substitution; Antiviral Agents; Drug Therapy, Combination; Evolution, Molecular; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Male; Middle Aged; Phylogeny; Retrospective Studies; Treatment Failure; Viral Envelope Proteins; Viral Load

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Cluster Analysis; Conserved Sequence; eIF-2 Kinase; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; HIV Infections; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Phosphorylation; Phylogeny; Ribavirin; Sequence Alignment; Sequence Analysis, DNA; Serum; Viral Envelope Proteins

Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture-produced HCV (HCVcc) and ex vivo-characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid-free subviral LVPs are assembled and secreted by apoB-producing cell lines. To determine whether such subviral particles circulate in HCV-infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low-density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A-purified LVPs contained at least the envelope glycoprotein E2 and E2-specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride-rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 10(6) apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid-free, envelope glycoprotein-containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes.. LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family.

Topics: Adult; Aged; Apolipoproteins B; Blotting, Western; Cohort Studies; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Lipoproteins, HDL; Lipoproteins, IDL; Lipoproteins, LDL; Male; Middle Aged; Nucleocapsid Proteins; Prognosis; Regression Analysis; RNA, Viral; Sensitivity and Specificity; Viral Envelope Proteins; Viral Load

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.

Topics: Adult; Aged; Cluster Analysis; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Transplantation; Longitudinal Studies; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load

The rate of non-response to pegylated interferon plus ribavirin (peg-IFN+RBV) in HCV/HIV coinfected patients is higher than in HCV-monoinfected patients. In this sense, the contribution of HCV genetic variability is unknown. The 5' untranslated (5'UTR), the nonstructural 5A (NS5A) and the second envelope (E2) HCV genomic regions have been implicated to peg-IFN therapy response. The proteins appear to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and the 5'UTR may influence the viral lymphotropism.. We examined comparatively the pretreatment HCV variability between HIV coinfected and HCV monoinfected patients as well as assessed longitudinally the impact of peg-IFN+RBV on HCV variability when HIV is co-present. For this purpose, 15 HIV coinfected and 20 HCV monoinfected patients were compared. They were peg-IFN+RBV non-responders and infected with HCV 1a.. Irrespectively of the HIV-coexistence, at baseline the amino acid variation in the NS5A-related domains was significantly higher than in the E2-PePHD (p<0.001). The number of amino acid variations (mean±SD) at the NS5A-ISDR domain was higher among HCV/HIV patients than HCV-monoinfected ones (1.80±0.77 vs. 0.95±1.05; p=0.009) but such difference was slightly lower when comparing NS5A-PKRBD sequences (2.47±1.13 vs. 1.60±1.57; p=0.06). No differences were found at the E2-PePHD (0±0 vs. 0.2±0.4). At intra-HIV coinfected patient level, only minor (HCV genetic analysis) or no (HCV substitution rate and quasispecies heterogeneity) changes were observed during therapy (basal, 24h, 4weeks, and 12weeks).. Among HCV-1a/HIV coinfected and HCV-monoinfected peg-IFN+RBV non-responder patients, the HCV variability at the 5'UTR, E2-PePHD and NS5A-PKRBD/ISDR domains was mostly comparable exhibiting a low number of variations. Four well-defined amino acid substitutions in NS5A-ISDR domain appeared most frequently when HIV coexists. The interferon-based therapy did not exert any effect in the variation, selection or diversity in the above mentioned HCV regions that could influence clinical responsiveness to IFN therapy.

Topics: 5' Untranslated Regions; Adult; Amino Acid Sequence; Antiviral Agents; Base Sequence; Drug Resistance, Viral; Female; Hepacivirus; Hepatitis C, Chronic; HIV Infections; Humans; Interferons; Male; Middle Aged; Molecular Sequence Data; Ribavirin; Sequence Alignment; Treatment Failure; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Cell Line; Disease Models, Animal; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Liver Transplantation; Mutation; Neutralization Tests; Pan troglodytes; RNA, Viral; Tetraspanin 28; Viral Envelope Proteins; Viral Load

The aim of this study was to investigate the association of mutations in the E2/NS1 [hypervariable regions 1 and 2 (HVR1 and HVR2)] and NS5A regions of the hepatitis C virus (HCV) genome and the effectiveness of interferon (IFN) therapy, and assess whether the degree of heterogeneity of HCV quasispecies predicts response to IFN treatment. Fourteen patients infected with HCV genotype 1b (HCV-1b) who were treated with pegylated IFN-α-2a and ribavirin for 24 weeks, were studied. E2/NS1 and NS5A gene segments were amplified by reverse-transcription polymerase chain reaction. HCV quasispecies heterogeneity in the E2/NS1 region was determined by cloning and sequencing. Mutations in the NS5A region were detected by direct sequencing. The heterogeneity of HCV quasispecies in the HVR1 was significantly greater in the non-responder group than in the responder group, but was not significant for HVR2 or NS5A. The correlation between mutations in IFN sensitivity-determining region (ISDR, NS5A2209-2248) and IFN sensitivity could not be supported. The degree of quasispecies heterogeneity in HVR1, but not in HVR2 and NS5A, may be predictive of response to IFN therapy. An ISDR may not apply to patients infected with HCV-1b.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Drug Therapy, Combination; Female; Genetic Association Studies; Genetic Variation; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Mutation, Missense; Polyethylene Glycols; Recombinant Proteins; Ribavirin; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins; Young Adult

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Amino Acids, Basic; Antibodies, Neutralizing; Antibodies, Viral; Binding Sites; Cell Line; Hepacivirus; Heparin; Hepatitis C, Chronic; Host-Pathogen Interactions; Humans; Immunoglobulin G; Lipoproteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Scavenger Receptors, Class B; Viral Envelope Proteins; Virus Internalization; Virus Replication

Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/B) serum levels in patients with different outcomes of HCV infection.. IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene.. In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p<0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p=0.005) and patients with spontaneously resolved hepatitis (p=0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs.. Our data suggest that HCV proteins modify IFN-lambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C.

Topics: Adult; Aged; Antigens, CD; Cross-Sectional Studies; Dendritic Cells; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferons; Interleukins; Male; Middle Aged; Polymorphism, Genetic; Prognosis; Tetraspanin 28; Toll-Like Receptor 2; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

Hepatitis C virus infections proceed to chronicity in the majority of cases. In patients, hepatitis C viruses exist as a dynamic and complex quasispecies. The dominant species at any one time arises in response to host immune pressure and other, incompletely understood factors. It is critical to understand all the mechanisms by which dominance is achieved, but this is difficult to study in vivo. Therefore, it would be useful to develop a cell culture system in which naturally occurring quasispecies could be studied. Hepatitis C virus glycoprotein genes E1 and E2 were PCR amplified as a cassette from the plasma of a chronically infected patient and shotgun cloned into a modified 1a/JFH1 infectious cDNA clone. Following transformation of bacteria, plasmids were batch harvested, transcribed, and transfected into Huh7.5 cells to produce a quasispecies of hypervariable region 1 (HVR1) that mimicked that circulating in vivo. Serial passage of the quasispecies in vitro resulted in replacement of the initially dominant species with a new HVR1 species coexisting with selected growth-enhancing mutations located outside HVR1. Antibody raised against one HVR1 sequence neutralized virus with the homologous HVR1 and cross-neutralized virus with a different sequence. Reciprocal swapping of the HVR1 regions between the two dominating species demonstrated that the HVR1 sequence affects the efficiency of replication and of neutralization by anti-HVR1 but that both processes are strongly influenced by regions outside HVR1.

Topics: Antibodies, Neutralizing; Cell Culture Techniques; Cell Line; Cloning, Molecular; Cross Reactions; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Hepatocytes; Humans; Mutation; Recombination, Genetic; Serial Passage; Transfection; Viral Envelope Proteins; Virus Replication

Hepatitis C virus roots a chronic liver disease. Currently approved treatment strategy includes administration of alpha interferon and ribavirin combined therapy for 24-48 weeks. One of the predictor of sustained virological response is an early virological response to treatment characterized as rapid response. Hyper variable region 1 (HVR1) of E2 protein is responsible for viral entry and acts as a target for neutralizing antibodies. Any mutation in this region would effect virus interaction with target cell and viral persistence.. Thirty one clones of six pre-treatment samples subjected to combination therapy were investigated. Three of the patients were rapid responders (R1, R2 and R3) and two were breakthrough responders (BT1 and BT2). Envelope 2 gene was amplified, cloned and sequenced. Amino acid substitution, frequency, composition and antigenic properties of HVR 1 of E2 protein were studied.. In both rapid responders (R.R) (14 amino acid sites) and breakthrough responders (BT.R) (13 amino acid sites) half of the amino acid sites were either conserved or resistant to any physiochemical change due to amino acid substitution. It also indicated that average composition of hydrophilic and basic amino acids were comparatively lower in rapid responders than other samples affecting probable interaction of virus with target cells. A central non antigenic region was constant among the breakthrough responders but differed in length significantly among rapid responders reflecting the adaptive nature of HVR1 to the immune response.. We observed that although HVR1is quite variable region in HCV 3a patients responding differently to treatment it still maintains its physiochemical properties for its proper functioning and viability.

Topics: Amino Acid Substitution; Antigens, Viral; Antiviral Agents; Drug Therapy, Combination; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Molecular Sequence Data; Polymorphism, Genetic; Ribavirin; RNA, Viral; Sequence Analysis, DNA; Treatment Outcome; Viral Envelope Proteins; Viral Load

Heterogeneity of subgenomic regions of hepatitis C virus (HCV) may be associated with response to interferon (IFN) therapy. The amino acid sequences of the PKR/eIF-2α phosphorylation homology domain (pePHD), IFN sensitivity determining region (ISDR), PKR binding domain (PKRBD), and variable region 3 (V3) were studied in 19 patients before and after 4 weeks of treatment. All patients were infected with HCV genotype 1a and were treated with pegylated-IFN and ribavirin. Thirteen patients achieved sustained viral response (responders) and six failed to clear viral RNA (nonresponders). The amino acid sequences in the pePHD and ISDR were identical in responders and nonresponders. However, amino acid substitution at position 2252 of PKRBD was significantly different between responders and nonresponders (P = 0.044). A larger number of mutations were observed in the V3 region of responders (P < 0.001). In this region, the amino acid in position 2364 differed between responders and nonresponders (responders: aspartic acid and serine, nonresponders: asparagine, P = 0.018). The amino acid sequences in the regions which were studied did not change after 4 weeks of treatment. It is concluded that the presence of specific amino acids in position 2252 of PKRBD and position 2364 of V3 might be associated with clinical response to IFN.

Topics: Adult; Amino Acid Substitution; Antiviral Agents; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Mutation, Missense; Polyethylene Glycols; Polymorphism, Genetic; Recombinant Proteins; Ribavirin; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins

Genetic heterogeneity is a characteristic feature of hepatitis C virus (HCV) and it reflects selection mechanisms affecting the virus. It is considered a major factor contributing the viral persistence and drug resistance. The following work presents the preliminary results of 5'UTR and E2/HVR1 genetic variation analysis and comparison in serum and peripheral blood mononuclear cells (PBMC) of 7 patients before and during the early phase of pegylated interferon alfa (PEG-IFN-alpha) and ribavirin therapy. Single strand conformational polymorphism (SSCP) analysis revealed genetic stability of 5'UTR in all but one patient who did not respond to treatment (SVR-), where new genetic variants appeared. E2/HVR1 genetic changes were characteristic in patients displaying treatment failure (SVR-) and usually reflected fluctuations in complexity and appearance of new genetic HCV variants. Genetic changes (reduction in complexity) were found in one of three sustained virological responders (SVR+ patients). Comparatory analysis of the HCV quasispecies present in serum and PBMC showed differences in at least one analysed region in all non-responders. Presented results suggest the independent forces driving genetic changes in analysed regions. They also point out the presence of genetic compartmentalization possibly having an impact on antiviral treatment result.

Topics: Antiviral Agents; Drug Therapy, Combination; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Poland; Polymorphism, Single-Stranded Conformational; Recombinant Proteins; Ribavirin; Sequence Alignment; Viral Envelope Proteins

Human and animal model evidence suggests that CD4+ T cells play a critical role in the control of chronic hepatitis C virus (HCV) infection. However, despite their importance, the mechanism behind the failure of such populations in chronic disease is not understood and the contribution of viral mutation is not known. To address this, this study defined the specificity and virological footprint of CD4+ T cells in chronic infection. CD8+ T-cell-depleted peripheral blood mononuclear cells from 61 HCV genotype 1-infected patients were analysed against a panel of peptides covering the HCV genotype 1 core--a region where CD4+ T-cell responses may be reproducibly obtained. In parallel, the core region and E2 protein were sequenced. Gamma interferon-secreting CD4+ T-cell responses directed against multiple epitopes were detected in 53% of individuals, targeting between one and four peptides in the HCV core. Viral sequence evaluation revealed that these CD4+ T-cell responses were associated with mutants in 2/21 individuals. In these two cases, the circulating sequence variant was poorly recognized by host CD4+ T cells. Bioinformatics analyses revealed no overall evidence of selection in the target epitopes and no differences between the groups with and without detectable CD4+ T-cell responses. It was concluded that sustained core peptide-specific CD4+ T-cell responses may be reproducibly measured during chronic HCV infection and that immune escape may occur in specific instances. However, overall the virological impact of such responses is limited and other causes for CD4+ T-cell failure in HCV must be sought.

Topics: Adult; Aged; Animals; CD4-Positive T-Lymphocytes; Epitopes; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-gamma; Lymphocyte Depletion; Male; Middle Aged; Molecular Sequence Data; RNA, Viral; Sequence Analysis, DNA; Viral Core Proteins; Viral Envelope Proteins

Hepatitis C virus (HCV) is a major cause of liver disease throughout the world. The NS5A and E2 proteins of HCV genotype 1 were reported to inhibit the double-stranded (ds) RNA-dependent protein kinase (PKR), which is involved in the cellular antiviral response induced by interferon (IFN). The response to IFN therapy is quite different between genotypes, with response rates among patients infected with types 2 and 3 that are two-three-fold higher than in patients infected with type 1. Interestingly, a significant percentage of HCV genotype 3-infected patients do not respond to treatment at all. The aim of this paper was to analyse the sequences of fragments of the E2 and NS5A regions from 33 outpatients infected with genotype 3a, including patients that have responded (SVR) or not responded (NR) to treatment. HCV RNA was extracted and amplified with specific primers for the NS5A and E2 regions and the PCR products were then sequenced. The sequences obtained covered amino acids (aa) 636-708 in E2 and in NS5A [including the IFN sensitivity determining region (ISDR), PKR-binding domain and extended V3 region)]. In the E2 and NS5A regions, we did observe aa changes among patients, but these changes were not statistically significant between the SVR and NR groups. In conclusion, our results suggest that the ISDR domain is not predictive of treatment success in patients infected with HCV genotype 3a.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Molecular Sequence Data; Polymerase Chain Reaction; Ribavirin; RNA, Viral; Viral Envelope Proteins; Viral Nonstructural Proteins

To elucidate adaptive evolution patterns and perform a positive selection analysis of hepatitis C virus(HCV) genotype 1b envelope 2 gene(E2) during natural chronic infection by a longitudinal study.. HCV E2 quasispecies profiles were derived from partially sequenced domains of 6 000 bp recombinant clones. Phylogenetic trees for HCV E2 gene were constructed by MEGA software and the specific codons undergoing diversifying positive selection were identified by FEL method.. HCV phylogenies, coupled with the number and distribution of selected sites were differed markedly between patients. HCV quasispecies complexity during chronic infection was not associated with the evolutionary time and the dominant viriant alternation of HCV quasispecies may occur after more than six months apart. Five sites under positive selection were identified within the ectodomain of the E2 protein.. A series of serum specimens for studies based on dominant viriant of HCV dynamic quasispecies is recommended to be collected at every six months. Several individual sites of HCV E2 gene are under a strong host immune pressure, position aa384, aa399 and aa410 may be involved in escape from neutralizing antibodies, while position aa475, aa522 may correlate to modulate the virus-receptor interaction which result in evading immunity.

Topics: Amino Acid Sequence; Evolution, Molecular; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Phylogeny; Sequence Homology, Amino Acid; Viral Envelope Proteins

HCV (hepatitis C virus) infection is among the leading causes of chronic liver disease, but currently there is no vaccine available. Data have accumulated about the importance of targeting different HCV antigens in vaccine candidate preparations. Here, a surface response study to select the optimal ratio of recombinant HCV structural antigens in a vaccine preparation, capable of generating in vivo functional cellular immune response in mice, was performed. The immunogenicity of the selected HCV structural protein mixture (Co-E1-E2) in mice and African green monkeys, after five doses of immunization, was also demonstrated. Specific T-cell proliferative response against HCV structural antigens was induced in vaccinated mice. Moreover, on challenge with recombinant HCV VV (vaccinia virus), all mice controlled the viraemia and 80% were protected. On the other hand, monkeys immunized with Co-E1-E2 developed antibodies, specifically directed to region 412-438 of E2 protein, that include an epitope implicated in HCV neutralization, in addition to a specific proliferative response against HCV Core and E2 proteins. These results indicated that the optimal amount and ratio of HCV recombinant proteins should be taken into account to elicit a successful immune response against HCV and therefore have important implications for vaccine design.

Topics: Animals; Chlorocebus aethiops; Female; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Immunity, Cellular; Male; Mice; Mice, Inbred BALB C; Recombinant Proteins; Viral Core Proteins; Viral Envelope Proteins; Viral Hepatitis Vaccines

Accumulating evidence suggests that certain features of hepatitis C virus (HCV), especially its high genetic variability, might be responsible for the low efficiency of anti-HCV treatment. Here, we present a bioinformatic analysis of HCV-1a populations isolated from 23 children with chronic hepatitis C (CHC) subjected to interferon-ribavirin therapy. The structures of the viral quasispecies were established based on a 132-amino-acid sequence derived from E1/E2 protein, including hypervariable region 1 (HVR1). Two types of HCV populations were identified. The first type, found in non-responders, contained a small number of closely related variants. The second type, characteristic for sustained responders, was composed of a large number of distantly associated equal-rank variants. Comparison of 445 HVR1 sequences showed that a significant number of variants present in non-responding patients are closely related, suggesting that certain, still unidentified properties of the pathogen may be key factors determining the result of CHC treatment.

Topics: Adolescent; Antiviral Agents; Child; Cluster Analysis; DNA, Viral; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Polymorphism, Genetic; Recombinant Proteins; Ribavirin; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins

Hepatitis C virus (HCV) can persist in the liver, lymphoid cells, and serum of individuals with apparently complete spontaneous or therapy-induced resolution of hepatitis C and can replicate in vivo and in vitro in human T cells. The current study was aimed at assessing the infectivity of HCV persisting at very low levels using the previously established HCV infection system in human T cells. Naive lymphoid cells were exposed to plasma and/or supernatants from cultured peripheral blood mononuclear cells from nine individuals with apparent sustained virological response after completion of antiviral therapy. Exposed cells were analyzed for HCV RNA-positive and HCV RNA-negative strands and, in selected cases, for HCV nonstructural protein 5a (NS5a), the appearance of HCV variants, and the release of virions by immunoelectron microscopy (IEM). The results showed that 11 of the 12 established cultures became HCV RNA-positive strand-reactive, whereas 4 also expressed the virus replicative strand. NS5a protein was detected in the de novo infected cells, and clonal sequencing revealed HCV variants not found in inocula. IEM demonstrated enveloped HCV particles in plasma used as inocula and in culture supernatant from T cells exposed to that plasma. Overall, HCV carried in three of the nine individuals studied elicited productive infection in vitro.. HCV persisting at very low levels long after therapy-induced resolution of chronic hepatitis C can remain infectious. The retained biological competence of the virus might have implications with respect to the mechanisms of its persistence and the epidemiology of HCV infection.

Topics: Adult; Antibodies, Monoclonal; Antigens, CD; Antiviral Agents; Cells, Cultured; Female; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Leukocytes, Mononuclear; Male; Middle Aged; Ribavirin; RNA, Viral; T-Lymphocytes; Tetraspanin 28; Viral Envelope Proteins; Viral Nonstructural Proteins

The molecular mechanisms of lymphoproliferation associated with the disruption of interferon (IFN) signaling and chronic hepatitis C virus (HCV) infection are poorly understood. Lymphomas are extrahepatic manifestations of HCV infection; we sought to clarify the molecular mechanisms of these processes.. We established interferon regulatory factor-1-null (irf-1(-/-)) mice with inducible and persistent expression of HCV structural proteins (irf-1/CN2 mice). All the mice (n = 900) were observed for at least 600 days after Cre/loxP switching. Histologic analyses, as well as analyses of lymphoproliferation, sensitivity to Fas-induced apoptosis, colony formation, and cytokine production, were performed. Proteins associated with these processes were also assessed.. Irf-1/CN2 mice had extremely high incidences of lymphomas and lymphoproliferative disorders and displayed increased mortality. Disruption of irf-1 reduced the sensitivity to Fas-induced apoptosis and decreased the levels of caspases-3/7 and caspase-9 messenger RNA species and enzymatic activities. Furthermore, the irf-1/CN2 mice showed decreased activation of caspases-3/7 and caspase-9 and increased levels of interleukin (IL)-2, IL-10, and Bcl-2, as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes. IL-2 and IL-10 were induced by the HCV core protein in splenocytes.. Disruption of IFN signaling resulted in development of lymphoma, indicating that differential signaling occurs in lymphocytes compared with liver. This mouse model, in which HCV expression and disruption of IFN signaling synergize to promote lymphoproliferation, will be an important tool for the development of therapeutic agents that target the lymphoproliferative pathway.

Topics: Age Factors; Animals; Apoptosis; B-Lymphocytes; Caspases; Cell Proliferation; Disease Models, Animal; fas Receptor; Female; Hepacivirus; Hepatitis C, Chronic; Interferon Regulatory Factor-1; Interleukin-10; Interleukin-12; Interleukin-2; Interleukins; Lymphoma; Lymphoproliferative Disorders; Male; Mice; Mice, Knockout; Mice, Transgenic; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Spleen; T-Lymphocytes; Time Factors; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412-426 (epitope I) and 434-446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.

Topics: Amino Acid Sequence; Animals; Epitopes; Genotype; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Neutralization Tests; Pan troglodytes; Vaccination; Viral Envelope Proteins; Viral Hepatitis Vaccines

Viral genome analyses performed in adult HCV-patients yielded very inconsistent results and are not transferable to children who are often infected vertically during a state of high immune tolerance. We analysed the mutational frequency in the PKR-binding domain (PKR-BD) of NS5A and PePHD of E2 protein pre- and post-treatment with peginterferon-alfa-2b and ribavirin in children chronically infected with HCV genotype 1. Amino acid sequences of NS5A (2 209-2 274) and E2 (618-681) were determined in serum samples using standard PCR procedures. Concerning the PKR-BD a significant higher number of mutations was observed in vertically compared to horizontally infected patients (2.14 vs 1.24, P-value = 0.03). This difference was exclusively based on the increased number of mutations in responders vs non-responders in vertically infected patients (2.95 vs 1.33; P-value = 0.02). While all patients with at least four mutations (n = 3) did respond to therapy, no other predictive parameters could be identified. In the PePHD no differences could be observed between either of these groups. These findings support the idea that viral properties, mode and therewith time of infection in terms of immune tolerance are equally important factors for predicting SVR in children. However given the low number of cases further studies are required to confirm this hypothesis.

Topics: Adolescent; Amino Acid Sequence; Antiviral Agents; Child; Child, Preschool; Disease Transmission, Infectious; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Infectious Disease Transmission, Vertical; Interferon alpha-2; Interferon-alpha; Male; Molecular Sequence Data; Mutation, Missense; Polyethylene Glycols; Polymerase Chain Reaction; Recombinant Proteins; Ribavirin; Sequence Analysis, DNA; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins; Young Adult

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis worldwide. Viral attachment and entry, representing the first steps of virus-host cell interactions, are major targets of adaptive host cell defenses. The mechanisms of antibody-mediated neutralization by host neutralizing responses in HCV infection are only poorly understood. Retroviral HCV pseudotypes (HCVpp) and recombinant cell culture-derived HCV (HCVcc) have been successfully used to study viral entry and antibody-mediated neutralization.. In this study, we used these model systems to investigate the mechanism of antibody-mediated neutralization by monoclonal antienvelope antibodies and polyclonal anti-HCV immunoglobulins purified from HCV-infected patients.. Using a panel of monoclonal antienvelope antibodies, we identified an epitope within the E1 glycoprotein targeted by human neutralizing antibodies during postbinding events. Interestingly, we observed that host neutralizing responses in the majority of HCV-infected individuals include antibodies targeting HCV entry after binding of the virus to the target cell membrane. Using a kinetic assay based on HCVpp and HCVcc entry, we demonstrate that purified antiviral immunoglobulins derived from individual HCV-infected patients appear to inhibit HCV infection at an entry step closely linked to CD81 and scavenger receptor BI (SR-BI).. Our results indicate that host neutralizing responses in HCV-infected patients target viral entry after HCV binding most likely related to HCV-CD81, and HCV-SR-BI interactions, as well as membrane fusion. These findings have implications not only for the understanding of the pathogenesis of HCV infection but also for the design of novel immunotherapeutic and preventive strategies.

Topics: Adult; Aged; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antigens, CD; Cells, Cultured; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Immunoglobulin G; Membrane Fusion; Middle Aged; Receptors, Virus; Scavenger Receptors, Class B; Tetraspanin 28; Viral Envelope Proteins

In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.

Topics: Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Neutralization Tests; Viral Envelope Proteins; Virion

Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, provide a target for host immune recognition. HCV genotypes are classified into six genetic groups. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the correlation between antibody level and viral load, genotype, disease severity and response to treatment was investigated. The levels of antibodies to HCV glycoproteins E1 and E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. Reactivity against UKN3A1.28 in sera from patients infected with genotype 3 was significantly higher than corresponding reactivity between patients infected with genotype 1 and H77c. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. The pre-treatment level of anti-E1E2 was higher in sustained responders to combination therapy. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.

Topics: Antibodies, Viral; Antiviral Agents; Enzyme-Linked Immunosorbent Assay; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Cirrhosis; Severity of Illness Index; Statistics as Topic; Treatment Outcome; Viral Envelope Proteins; Viral Load

Hepatitis C virus (HCV) envelope glycoprotein co-evolution was studied in 14 genotype 1-infected and treatment-naive subjects, including 7 with mild and 7 with severe liver disease. Cassettes encoding the envelope 1 gene (E1) and hypervariable region (HVR1) of the envelope 2 gene were isolated at 38 different time points over 81 follow-up years. There were no significant differences in age, gender, alcohol use, or viral load between the mild and severe disease groups. Virus from subjects with severe disease had significantly slower evolution in HVR1, and significant divergent evolution of E1 quasispecies, characterized by a preponderance of synonymous mutations, compared to virus from subjects with mild disease. Phylogenetic comparisons indicated higher similarity between amino acid sequences of the E1 and HVR1 regions with mild disease versus severe disease (r=0.44 versus r=0.17, respectively; P=0.01). In summary, HCV envelope quasispecies co-evolution differs during mild versus severe disease.

Topics: Amino Acid Sequence; Evolution, Molecular; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Sequence Alignment; Viral Envelope Proteins

Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

Topics: Antiviral Agents; Drug Resistance, Viral; Genetic Variation; Haplotypes; Hepacivirus; Hepatitis C, Chronic; Humans; Interferons; Mutation, Missense; Ribavirin; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins

It is considered that selection pressure exerted by the host immune response during early HCV infection might influence the outcome of that infection particularly as it relates to persistence or clearance of the agent. However, it is unclear whether positive selection pressure plays a role in determining the severity of hepatitis C during the course of persistent HCV infection. To address the evolutionary mechanism by which HCV escapes from the host immune response and to assess the relationship between viral evolution and hepatic inflammation, we determined 57 sequences (3-5 serial samples per patient) from 5 individuals with persistent HCV infection of genotype 1a who were under long-term follow-up ranging from 15.6 to 21.6 years. We applied a novel method to estimate serial alternations of selective pressure against the HCV enveloped region and compared this to fluctuation in transaminase level over time. Positive selection pressure was reduced over time postinfection, as evidenced by a reduction in nonsynonymous substitutions in the later phase of infection. Furthermore, serum transaminase, as a measure of inflammatory necrosis of hepatocytes, was reduced in parallel with decreased positive selection pressure. These results suggest that during persistent HCV infection, the virus faces diminished immune pressure over time, either from mutation to an immune resistant sequence or from immunologic exhaustion, and that this diminished immune attack is reflected in diminished inflammatory activity. This observation may be applicable to other viruses characterized by a slow rate of disease progression.

Topics: Aged; Aged, 80 and over; Alanine Transaminase; Epitopes, B-Lymphocyte; Evolution, Molecular; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Inflammation; Liver; Male; Middle Aged; Phylogeny; Selection, Genetic; Time Factors; Viral Envelope Proteins

Using both a mass spectrometry-based method and the classical method of cloning and sequencing, we demonstrated weekly changes in the hypervariable region 1 quasispecies of a chimpanzee infected with an infectious clone, coinciding with neutralizing antibody emergence. We also used the mass spectrometry method in the clinical follow-up of a chronically infected patient over a 5-year period.

Topics: Amino Acid Sequence; Animals; Ape Diseases; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Mutation; Pan troglodytes; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Viral Envelope Proteins

Investigation of the mechanisms underlying hepatitis C virus (HCV) envelope glycoprotein gene evolution will greatly assist rational development of broadly neutralizing antibody-based vaccines or vaccine components. Previously, comprehensive cross-genotype evolutionary studies of E1E2 have not been possible due to the paucity of full-length envelope gene sequences representative of all major HCV genotypes (1-6) deposited in international sequence databases. To address this shortfall, a full-length E1E2 clone panel, corresponding to genotypes of HCV that were previously under-represented, was generated. This panel, coupled with divergent isolates available via international sequence databases, was subjected to high-resolution methods for determining codon-substitution patterns, enabling a fine-scale dissection of the selective pressures operating on HCV E1E2. Whilst no evidence for positive selection was observed in E1, the E2 protein contained a number of sites under strong positive selection. A high proportion of these sites were located within the first hypervariable region (HVR1), and statistical analysis revealed that cross-genotype adaptive mutations were restricted to a subset of homologous positions within this region. Importantly, downstream of HVR1, a differential genotype-specific distribution of adaptive mutations was observed, suggesting that subtly different evolutionary pressures shape present-day genotype diversity in E2 outside HVR1. Despite these observations, it is demonstrated that purifying selection due to functional constraint is the major evolutionary force acting on HCV E1E2. These findings are important in the context of neutralizing-antibody vaccine targeting, as well as in contributing to our understanding of E1E2 function.

Topics: Amino Acid Sequence; Genetic Variation; Genotype; Hepacivirus; Hepatitis C; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Phylogeny; Selection, Genetic; Sequence Analysis, DNA; Viral Envelope Proteins

Broadly reactive neutralizing antibodies (nAbs) and multispecific T-cell responses are generated during chronic hepatitis C virus (HCV) infection and yet fail to clear the virus. This study investigated the development of autologous nAb and HCV-glycoprotein-specific T-cell responses and their effects on viral sequence evolution during chronic infection in order to understand the reasons for their lack of effectiveness.. Numerous E1E2 sequences were amplified and sequenced from serum samples collected over a 26-year period from patient H, a uniquely well-characterized, chronically infected individual. HCV pseudoparticles (HCVpp) expressing the patient-derived glycoproteins were generated and tested for their sensitivity to neutralization by autologous and heterologous serum antibodies.. A strain-specific nAb response developed early in infection (8 weeks postinfection), whereas cross-reactive antibodies able to neutralize HCVpp-bearing heterologous glycoproteins developed late in infection (>33 wk postinfection). The humoral response continuously failed to neutralize viruses bearing autologous glycoprotein sequences that were present in the serum at a given time. The amplified glycoprotein sequences displayed high variability, particularly in regions corresponding to defined linear B-cell epitopes. Mutations in defined neutralizing epitopes were associated with a loss of recognition by monoclonal antibodies against these epitopes and with decreased neutralization of corresponding HCVpp. Viral escape from CD4 and CD8 T-cell responses also was shown for several novel epitopes throughout the glycoprotein region.. During chronic infection HCV is subjected to selection pressures from both humoral and cellular immunity, resulting in the continuous generation of escape variants.

Topics: Amino Acid Sequence; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cross Reactions; Epitopes, T-Lymphocyte; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunity, Cellular; Immunity, Innate; Molecular Sequence Data; Mutation; Neutralization Tests; Polymorphism, Genetic; RNA, Viral; T-Lymphocytes; Time Factors; Viral Envelope Proteins; Virion

The role of T-cells in clearance of hepatitis C virus (HCV) during acute infection is critical. The relevance of the immunological response in the control of HCV replication is less clear in chronic HCV infection. HCV-specific T-cell responses were examined in 92 interferon-naive individuals with chronic hepatitis C. A panel of 441 overlapping peptides spanning all expressed HCV proteins was used to measure HCV-specific T-cell responses, using flow cytometry after stimulating peripheral blood mononuclear cells (PBMCs) with different pools of these peptides. Most patients showed responses to at least one HCV protein, with NS5B for CD8(+) responses and E2 for CD4(+) responses identified most frequently. Both the prevalence and breadth of CD4(+) and CD8(+) responses were lower in co-infected patients, independently of the HCV genotype.

Topics: Adult; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Hepacivirus; Hepatitis C, Chronic; HIV; HIV Infections; Humans; Leukocytes, Mononuclear; Lymphocyte Activation; Middle Aged; Species Specificity; Viral Envelope Proteins; Viral Nonstructural Proteins

The role of neutralizing antibody (NAb) in determining response to antiviral therapy has not been established.. In this study we have analysed the kinetic's of the NAb response in patients with chronic hepatitis C who received antiviral therapy.. Seventeen patients infected with genotype 1, 2a/c or 3a hepatitis C virus (HCV) were enrolled, eight with a sustained virological response (SVR), five non-responders and four relapsers.. The mean NAb titre required to neutralize 50% of the E1E2-pp in patients who achieved an SVR (294+/-S.D. 51), in relapsers (246+/-S.D. 61.7) and non-responders (286+/-S.D. 80.95) did not differ significantly between the patient groups and did not alter during the course of treatment (P>0.01). Genetic variation present before antiviral therapy was analysed by single strand conformation polymorphism (SSCP) and failed to demonstrate a significant difference in the mean number of amplified E1E2 DNA fragments from the serum of patients who achieved an SVR (3.15+/-S.D. 1.53), relapsers (2.8+/-S.D. 1.32) or non-responders (3.69+/-S.D. 1.75). The baseline serum HCV viral loads were also not significantly different between patients who achieved an SVR (1.4 x 10(6) copies/ml; +/-S.D. 2.4 x 10(6)), relapsers (1.3 x 10(7) copies/ml; +/-S.D. 2.4 x 10(7)) and non-responders (1.5 x 10(6) copies/ml; +/-S.D. 1.1 x 10(6)).. We have shown that neutralizing anti-HCVpp antibody is not associated with response to antiviral therapy. In addition, there was no correlation between baseline virological load, circulating viral quasi-species, NAb titres and final response to treatment.

Topics: Antiviral Agents; Drug Therapy, Combination; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Interferons; Neutralization Tests; Polyethylene Glycols; Polymorphism, Single-Stranded Conformational; Ribavirin; Viral Envelope Proteins

End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period.. For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly.. HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT.. Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.

Topics: Adult; Amino Acid Sequence; Carcinoma, Hepatocellular; Cytokines; Evolution, Molecular; Female; Genetic Variation; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Cirrhosis; Liver Transplantation; Male; Middle Aged; Molecular Sequence Data; Viral Envelope Proteins; Viral Load

Hepatitis C virus (HCV) has a linear positive-stranded RNA genome of approximately 9,600 nucleotides in length and displays a high level of sequence diversity caused by high mutation rates and recombination. However, when we performed long distance reverse transcription-PCRs on HCV RNA isolated from serum of chronic HCV patients, not only full-length HCV genomes but also HCV RNAs which varied in size from 7,600 to 8,346 nucleotides and contained large in-frame deletions between E1 and NS2 were amplified. Carefully designed control experiments indicated that these deletion mutants are a bona fide natural RNA species, most likely packaged in virions. Moreover, deletion mutants were detected in sera of patients infected with different HCV genotypes. We observed that 7/37 (18.9%) of genotype 1, 5/43 (11.6%) of genotype 3, and 4/13 (30.7%) of genotype 6 samples contained HCV deletion mutant genomes. These observations further exemplify HCV's huge genetic diversity and warrant studies to explore their biological relevance.

Topics: Base Sequence; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Male; Molecular Sequence Data; Mutation; RNA, Viral; Sequence Analysis, RNA; Sequence Deletion; Viral Envelope Proteins

Ribavirin and interferon combination therapy is more effective than interferon monotherapy in patients with chronic hepatitis C virus (HCV) infection. To test the hypothesis that ribavirin induces nucleotide substitutions in the viral genome and reduces viral load by forcing it into error catastrophe in the combination therapy, we investigated the molecular evolution of HCV quasispecies in 3 patients who received combination therapy and 2 patients who received interferon monotherapy.. The quasispecies were analyzed before and after therapy by sequencing at least 8 clones in five regions of the HCV genome; 5' untranslated region, EI, E2, NS5A and NS5B.. Marked genetic drift was observed in the NS5A and NS5B regions in patients treated with combination therapy. However, genetic distances between clones obtained after therapy were closer than those obtained before therapy.. Our results suggest that the combination therapy modified HCV quasispecies, but that this did not reflect the induction of error catastrophe by ribavirin. Modification of quasispecies by this therapy requires further investigation in a larger number of patients to elucidate the possible mechanism of viral resistance against the combination therapy.

Topics: 5' Untranslated Regions; Antiviral Agents; Drug Therapy, Combination; Evolution, Molecular; Genetic Drift; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Mutation; Phylogeny; Recombinant Proteins; Ribavirin; Sequence Analysis, DNA; Sequence Homology; Viral Envelope Proteins; Viral Nonstructural Proteins

Dendritic cells (DC) are crucially involved in the induction of immune responses; however, reports on DC functions in chronic hepatitis C are controversial. Function of DC includes proper cell trafficking between sites of infection and lympho-cellular compartments. Thus, we analyzed DC compartmentalization and changes in DC migration in hepatitis C virus (HCV)-infected patients. We found significantly lower numbers of circulating BDCA1+ and BDCA2+ DC in HCV(+) patients (n = 20) than in healthy controls (n = 12) (P < .05). Analyzing liver samples from HCV(+) patients (n = 15), HCV(-) controls (n = 15), and disease controls (n = 10), we demonstrated chronic hepatitis C to be associated with intrahepatic DC enrichment (P < .05). In vitro studies indicated that HCV E2-induced secretion of RANTES efficiently attracts CCR5(+) immature DC. Incubation of DC with sera derived from HCV(+) patients made DC unresponsive to CCL21, the chemokine recruiting DC to lymphoid tissues for T cell priming. Unlike attraction of CCR5+ DCs via RANTES, direct inhibition of DC migration in response to CCL21 was specific for patients with chronic hepatitis C and could be attributed to interaction of HCV E2 with CD81 on DC. In conclusion, migration of DC is markedly affected by interaction of HCV E2 with CD81. Failure of DC to recirculate to lymphoid tissue may be critically involved in impaired T cell priming during HCV infection.

Topics: Adult; Aged; Antigens, CD; Antigens, Surface; Cell Movement; Chemokine CCL21; Chemokine CCL5; Chemokines, CC; Dendritic Cells; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Liver; Male; Middle Aged; Receptors, Virus; T-Lymphocytes; Tetraspanin 28; Viral Envelope Proteins

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.

Topics: Animals; Antigens, CD; Cells, Cultured; DNA; DNA-Binding Proteins; Hepatitis C, Chronic; Humans; Liver; Liver Cirrhosis; Matrix Metalloproteinase 2; Mice; NIH 3T3 Cells; Phosphorylation; Signal Transduction; Tetraspanin 28; Transcription Factor AP-2; Transcription Factors; Up-Regulation; Viral Envelope Proteins

A series of 29 patients undergoing treatment for chronic hepatitis C virus (HCV) genotype 1 infection with pegylated alpha-2a interferon plus ribavirin were studied for patterns of response to antiviral therapy and viral quasispecies evolution. All patients were treatment naive and had chronic inflammation and fibrosis on biopsy. As part of an analysis of pretreatment variables that might affect the outcome of treatment, genetic heterogeneity within the viral E1-E2 glycoprotein region (nucleotides 851 to 2280) was assessed by sequencing 10 to 15 quasispecies clones per patient from serum-derived PCR products. Genetic parameters were examined with respect to response to therapy based on serum viral RNA loads at 12 weeks (early viral response) and at 24 weeks posttreatment (sustained viral response). Nucleotide and amino acid quasispecies complexities of the hypervariable region 1 (HVR-1) were less in the responder group in comparison to the nonresponder group at 12 weeks, and genetic diversity was also less both within and outside of the HVR-1, with the difference being most pronounced for the non-HVR-1 region of E2. However, these genetic parameters did not distinguish responders from nonresponders for sustained viral responses. Follow-up studies of genetic heterogeneity based on the HVR-1 in selected responders and nonresponders while on therapy revealed greater evolutionary drift in the responder subgroup. The pretreatment population sequences for the NS5A interferon sensitivity determinant region were also analyzed for all patients, but no correlations were found between treatment response and any distinct genetic markers. These findings support previous studies indicating a high level of genetic heterogeneity among chronically infected HCV patients. One interpretation of these data is that early viral responses are governed to some extent by viral factors, whereas sustained responses may be more influenced by host factors, in addition to effects of viral complexity and diversity.

Topics: Adolescent; Adult; Aged; Amino Acid Sequence; Base Sequence; DNA, Viral; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Polyethylene Glycols; Recombinant Proteins; RNA, Viral; Sequence Homology, Amino Acid; Time Factors; Viral Envelope Proteins; Viral Nonstructural Proteins

The role of antibody dependent cellular cytotoxicity (ADCC) in HCV infection is unclear at present. Antibodies mediating ADCC are usually directed against viral envelope proteins. As cell surface expression of the HCV envelope E2 protein has been shown, the HCV E2 protein is an especially promising candidate target for ADCC.. Sera from patients with acute (n=6), self-limited (n=11) and chronic (n=19) HCV infection were analyzed in this study. Sera reacting with cell-bound HCV antigens were examined in a flowcytometric cytotoxicity assay using antigen-coated JOK-1 cells as targets.. We found that sera from all stages of HCV infection reacted with cells loaded with HCV E2. E2-specific ADCC was observed in patients with acute (n=3/6), self-limited (n=5/11) and chronic (n=13/19) hepatitis C and was closely related to fluorescence intensity in the E2-binding assay (r=0.67, P<0.001).. We conclude that E2-antibodies from all stages of HCV infection can mediate ADCC. Thus, the role of this process in the pathogenesis of chronic hepatitis C should be further elucidated.

Topics: Adolescent; Adult; Aged; Animals; Antibody-Dependent Cell Cytotoxicity; CHO Cells; Cricetinae; Female; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Male; Middle Aged; Recombinant Proteins; Viral Envelope Proteins; Viral Load

The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.

Topics: Animals; Antibodies, Viral; Apolipoprotein C-I; Apolipoproteins C; Cross Reactions; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Neutralization Tests; Pan troglodytes; Viral Envelope Proteins; Virulence

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.

Topics: Amino Acid Sequence; Egypt; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Epidemiology; Molecular Sequence Data; Phylogeny; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Nonstructural Proteins

B-cell lymphoproliferative disorders (BCLD) have been associated with chronic hepatitis C virus (HCV) infection. The HCV glycoprotein E2 (gpE2) hypervariable region I (HVR-I) may be a potential antigenic candidate to promote B-cell proliferation. The purpose of this study was to analyze the influence of HVR-I sequence variability in the development of BCLD. HVR-I sequences were studied in 29 chronically HCV-infected patients with (n=15) or without (n=14) BCLD. After PCR amplification of the gpE2 region, analysis of the 81 bp HVR-I encoding fragment was performed on 7-18 clones per patient. HVR-I sequence complexity was slightly lower in patients with BCLD (mean 0.347) than without (0.468) (P=0.2), though, sequence diversities were similar (0.0370 vs 0.0954, P=0.239). Phylogenetic analysis did not reveal any BCLD-associated clustering. In our population, neither the recently described insertion between positions 1 and 2 of HVR-I nor residues at positions 4 and 13 were particularly linked to BCLD. As previously described, we confirm the high degree of conservation of HVR-I residues T-2, G-6 and G-23 in our patients. Contrary to recent findings, our analysis based on multiple clones per patient analysis did not reveal any particular motif associated with BCLD.

Topics: Adult; Aged; Amino Acid Sequence; B-Lymphocytes; Cell Division; Female; Gene Rearrangement, B-Lymphocyte; Genetic Heterogeneity; Hepacivirus; Hepatitis C, Chronic; Humans; Lymphoproliferative Disorders; Male; Middle Aged; Molecular Sequence Data; Mutation; Phylogeny; Viral Envelope Proteins; Viral Proteins

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.

Topics: Centrifugation, Density Gradient; Hepacivirus; Hepatitis C, Chronic; Humans; Immunoprecipitation; Microscopy, Immunoelectron; RNA, Viral; Viral Core Proteins; Viral Envelope Proteins; Viremia

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are important targets for the host immune response. The genes encoding these proteins exhibit a high degree of variability that gives rise to differing phenotypic traits, including alterations in receptor-binding affinity and immune recognition and escape. In order to elucidate patterns of adaptive evolution during chronic infection, a panel of full-length E1E2 clones was generated from sequential serum samples obtained from four chronically infected individuals. By using likelihood-based methods for phylogenetic inference, the evolutionary dynamics of circulating HCV quasispecies populations were assessed and a site-by-site analysis of the d(N)/d(S) ratio was performed, to identify specific codons undergoing diversifying positive selection. HCV phylogenies, coupled with the number and distribution of selected sites, differed markedly between patients, highlighting that HCV evolution during chronic infection is a patient-specific phenomenon. This analysis shows that purifying selection is the major force acting on HCV populations in chronic infection. Whilst no significant evidence for positive selection was observed in E1, a number of sites under positive selection were identified within the ectodomain of the E2 protein. All of these sites were located in regions hypothesized to be exposed to the selective environment of the host, including a number of functionally defined domains that have been reported to be involved in immune evasion and receptor binding. Dated-tip methods for estimation of underlying HCV mutation rates were also applied to the data, enabling prediction of the most recent common ancestor for each patient's quasispecies.

Topics: Amino Acid Sequence; Evolution, Molecular; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Mutation; Phylogeny; Selection, Genetic; Sequence Analysis, DNA; Viral Envelope Proteins

In an effort to clarify the life cycle of HCV, the HCV genome in liver biopsies taken from chronic active hepatitis C patients undergoing interferon treatment was investigated. Molecular cloning by long distance reverse-transcription polymerase chain reaction (RT-PCR) revealed that the HCV genome in two patients with high viral loads in the liver had in-frame deletions of approximately 2 kb between E1 and NS2, which encode the E1-NS2 fusion protein and six other HCV proteins: core, NS3, NS4A, NS4B, NS5A, and NS5B. Among the remaining 21 chronic active hepatitis C patients, these types of deletion were found in another two patients and in two hepatocellular carcinoma patients. Out-of-frame deletions in the structural region were isolated from the other five patients, but the dominant RT-PCR products were non-truncated genomes. Retrospective analysis of a series of serum samples taken from a patient carrying the subgenome with the in-frame deletion revealed that both the subgenome and the full genome persisted through the 2-year period of investigation, with the subgenome being predominant during this period. Sequence analysis of the isolated cDNA suggested that both the subgenome and the full genome evolved independently. Western blotting analysis of HCV proteins from the HCV subgenome indicated that they were processed in the same way as those from the full genome. HCV subgenomes thus appear to be involved in the HCV life cycle.

Topics: Amino Acid Sequence; Base Sequence; Biopsy; Gene Deletion; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Liver; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins; Virus Replication

Hepatitis C virus (HCV) displays high genetic diversity. Inter-host sequence variability may mainly reflect a neutral drift evolution. In contrast, intra-host evolution may be driven by an adaptive selection to host responses to infection. Here, HCV E2 intra-host evolution in two patients during the course and follow-up of successive treatments with IFN-alpha and IFN-alpha/ribavirin was investigated. Phylogenetic analyses suggested that adaptive pressures prompt a continuous selection of viral variants derived from the previous ones (intra-lineage evolution) and/or a swapping of viral lineages during the course of the infection (inter-lineage evolution). Selection would act not only on the phenotypic features of hypervariable region 1 (HVR1) but also on those of the flanking regions. The pressures operate mainly at the amino acid level, but they also appeared to act on nucleotide sequences. Moreover, HVR1 heterogeneity seemed to be strongly constrained. This work contributes to the knowledge of HCV intra-host evolution during chronicity.

Topics: Antiviral Agents; DNA, Viral; Drug Therapy, Combination; Evolution, Molecular; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Molecular Sequence Data; Phylogeny; Recombinant Proteins; Ribavirin; Treatment Outcome; Viral Envelope Proteins

Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.

Topics: Adult; Aged; Cross Reactions; Cucumovirus; Epitopes; Female; Hepacivirus; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Male; Middle Aged; Mutagenesis, Insertional; Nicotiana; Plant Diseases; Viral Envelope Proteins

The effect of highly active antiretroviral therapy (HAART) on HCV replication is controversial, with some studies reporting no effect and others increases, reductions and even clearances of HCV RNA after treatment. In this study, the effect of HAART was investigated on the titre of anti-HCV specific antibodies and on the relationship between these antibodies and HCV RNA level in a cohort of 24 patients with inherited bleeding disorders. A significant inverse correlation between antibodies to both total HCV proteins and HCV RNA (R = -0.42, P = 0.05) and between antibodies to HCV envelope glycoproteins and HCV RNA (R = -0.54, P = 0.01) was observed pre-HAART. The relationship disappeared or was obscured after therapy (R = 0.24, P = 0.30 and R = 0.16, P = 0.50, respectively). Thus, we show that HAART affects the HCV specific humoral immune responses without affecting the HCV RNA level.

Topics: Animals; Antiretroviral Therapy, Highly Active; Cells, Cultured; Hemophilia A; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; HIV Infections; HIV-1; Humans; Male; RNA, Viral; Spodoptera; Viral Envelope Proteins; Viral Load; Viral Structural Proteins

The aim of this study was to determine whether specific sequences of the phosphorylation homology domain (PePHD) region could be correlated with differences in response to antiviral therapy in patients infected with hepatitis C virus subtypes 1b, 2c, 3a and 4c/d. We included 43 patients (22 sustained responders and 21 nonresponders or relapsers) in the study, who were classified according to early viral decline during the first weeks of antiviral treatment and response at end of follow up. Type of mutations, mutation frequency, genetic diversity and phylogenetic relationships were compared at the PePHD and flanking regions. Phylogenetic trees showed that each sequence clustered together with those of the same subtype. Sequences from subtypes 1b and 4c/d resembled more closely the phosphorylation sites of protein kinase R and eIF2 alpha than sequences from genotypes 2c and 3a, the latter with higher response rates to interferon-alpha (IFN alpha) treatment. However, within specific subtypes, no separate clusters of responders and nonresponders were observed either at the beginning or at the end of follow up. We were not able to find any particular sequence or mutation in the PePHD region or in any other subregion of the fragment studied that allowed prediction of treatment response.

Topics: Adult; Amino Acid Motifs; Amino Acid Sequence; Antiviral Agents; Drug Resistance, Viral; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Phosphorylation; Phylogeny; RNA, Viral; Sequence Alignment; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load

Antibodies against envelope glycoprotein 1 and 2 (anti-E1/E2) have been suggested to influence HCV replication levels. Hepatitis B virus (HBV) may interfere with hepatitis C virus (HCV) replication. At present there are no data on anti-E1/E2 antibody responses or on the effect of interferon (IFN) treatment in HBV-HCV co-infection. Accordingly, we evaluated serum anti-E1/E2 antibodies in 50 patients (median age, 26.5; males, 30) with chronic hepatitis, 38 with HCV and 12 with HBV-HCV co-infection, who had undergone alpha-IFN treatment. Before starting IFN, the HCV group showed higher HCV-RNA levels (bDNA assay) than the HBV-HCV group (median 3.75 vs. 0.64 x 10(6) Eq/ml, respectively; P < 0.05). Similarly, the anti-E2 levels (EIA assay) were higher in the HCV group than in the HBV-HCV (mean +/- SD, 53.8 +/- 54.58 vs. 24.5 +/- 41.50 U/ml, respectively; P < 0.02), and the prevalence of anti-E2 was also higher in the HCV group (94 vs. 58%, respectively; P < 0.007). No correlation was found between anti-E1/E2 antibodies and the HCV-RNA levels. The prevalence of E1/E2 antibodies was similar in the different HCV genotypes. Higher baseline levels of anti-E2 antibodies and a decrease or disappearance of anti-E2 antibodies during IFN were associated with IFN sustained response in both groups, whereas no reduction in the anti-E1/E2 levels was observed in non-responders. The data show that HBV co-infection influences both HCV replication and the anti-E1/E2 antibody production. High pre-treatment levels of anti-E2 antibodies and their decrease or disappearance during interferon treatment are often associated with HCV clearance in sustained responders, irrespective of the HCV genotype.

Topics: Adolescent; Adult; Base Sequence; Child; DNA, Viral; Female; Hepacivirus; Hepatitis B virus; Hepatitis B, Chronic; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Recombinant Proteins; RNA, Viral; Viral Envelope Proteins; Virus Replication

This study aimed to investigate the relation between hepatitis C virus (HCV) antibody profiles and response to therapy of chronic HCV infection of genotype 1b.. Quantitative assays of antibody response to HCV antigens were performed sequentially using immunoblot confirmatory assays in 25 patients with genotype 1b chronic hepatitis C who received a 24-week course of interferon and ribavirin therapy.. 12 patients had a sustained response (group A), and 13 did not (group B). Serum titers of HCV RNA were significantly higher in group B than in group A (p = 0.02). Pretreatment antibody reactivity to core, NS3, NS4, and NS5 antigens did not differ significantly between the two groups, but group A had significantly higher titers of anti-E2/NS1 than group B (p = 0.04). Sustained response was noted in none of seven patients with HCV RNA > 10(6) copies/ml, but did occur in 12 of 18 patients with HCV RNA < 10(6) copies/ml (p < 0.01). Among the latter, all seven patients with anti- E2/NS1 had sustained responses, as did five (45%) of 11 without (p = 0.04). Antibody profiles changed little or not at all at the end of treatment or at 6 months after treatment in both groups.. In chronic hepatitis C of genotype 1b, patients with HCV RNA > 10(6) copies/ml respond poorly to therapy compared to those with HCV RNA < 10(6) copies/ml. Among the latter, the presence of pretreatment anti-E2/NS1 correlated with sustained response. Quantitative assay of antibody response to HCV antigens at the end of treatment had no additional value to predict a sustained response.

Topics: Adult; Aged; Antiviral Agents; Drug Therapy, Combination; Female; Hepacivirus; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Middle Aged; Ribavirin; RNA, Viral; Viral Envelope Proteins; Viral Nonstructural Proteins

Little is known about the role of the humoral immune response to hepatitis C virus (HCV). This study provides molecular evidence for the mechanism by which neutralizing Abs from the sera of chronic HCV patients have lower inhibitory activities against the binding of HCV E2 envelope protein to human hepatoma cell lines than to a lymphoma cell line. E2 binds to several putative receptors, specifically human CD81; human scavenger receptor, class B, type 1; and heparan sulfate. We have shown that E2 binds to target cells via these receptors in a noncompetitive manner. Thus, incomplete inhibition of one of the receptors leads to only a partial E2 blockade and, possibly, evasion of the host immune response. We demonstrated that the difference in and reduction of inhibition was closely related to impaired blockade of E2 binding to scavenger receptor, class B, type 1, and heparan sulfate. We have also shown that soluble E2 protein binds to multiple soluble receptors via separate binding domains on E2, providing further evidence for the distinct recognition of multiple cellular receptors by E2. This report suggests a novel finding that biased humoral immune responses to HCV E2 might provide an alternative mechanism for viral escape without the involvement of mutation. Additionally, our data give crucial consideration to the development of HCV vaccines that stimulate protective humoral immune responses.

Topics: Antigens, CD; Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Receptors, LDL; Receptors, Virus; Tetraspanin 28; Viral Envelope Proteins

Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.

Topics: Blotting, Western; Enzyme-Linked Immunosorbent Assay; Hepacivirus; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Recombinant Proteins; Renal Dialysis; RNA, Viral; Viral Envelope Proteins; Viral Load; Viremia

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

Topics: Adult; Aged; Amino Acid Sequence; Antigens, CD; Binding Sites; CD8-Positive T-Lymphocytes; Female; Flow Cytometry; Gene Expression; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Mutation; Protein Binding; Receptors, Virus; Sequence Analysis; Tetraspanin 28; Viral Envelope Proteins

Little is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied nine chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as five responders and five non-responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD, and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non-responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.

Topics: Adult; Amino Acid Substitution; Antiviral Agents; Capsid Proteins; Drug Resistance, Viral; Drug Therapy, Combination; Female; Genetic Variation; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Interferons; Male; Middle Aged; Mutation; Phylogeny; Polymorphism, Single-Stranded Conformational; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; RNA, Viral; Selection, Genetic; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

Previous work on hepatitis C virus (HCV) led to the discovery of a new form of virus particle associating virus and lipoprotein elements. These hybrid particles (LVP for lipo-viro-particles) are enriched in triglycerides and contain at least apolipoprotein B (apoB), HCV RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine, the two main organs specialized in the production of apoB-containing lipoprotein. To identify the site of LVP production, the genetic diversity and phylogenetic relationship of HCV quasispecies from purified LVP, whole serum and liver biopsies from chronically infected patients were studied. HCV quasispecies from LVP and liver differed significantly, suggesting that LVP were not predominantly synthesized in the liver but might also originate in the intestine. The authors therefore searched for the presence of HCV in the small intestine. Paraffin-embedded intestinal biopsies from 10 chronically HCV-infected patients and from 12 HCV RNA-negative controls (10 anti-HCV antibody-negative and two anti-HCV antibody-positive patients) were tested for HCV protein expression. HCV NS3 and NS5A proteins were stained in small intestine epithelial cells in four of the 10 chronically infected patients, and not in controls. Cells expressing HCV proteins were apoB-producing enterocytes but not mucus-secreting cells. These data indicate that the small intestine can be infected by HCV, and identify this organ as a potential reservoir and replication site. This further emphasizes the interaction between lipoprotein metabolism and HCV, and offers new insights into hepatitis C infection and pathophysiology.

Topics: Adult; Apolipoproteins B; Biopsy; Epithelial Cells; Genetic Variation; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Immunohistochemistry; Intestine, Small; Liver; Membrane Proteins; Molecular Sequence Data; Phylogeny; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

Two chimpanzees, 1535 and 1536, became persistently infected following inoculation with RNA transcripts from cDNA clones of hepatitis C virus (HCV). Analysis of the HCV genomes from both animals showed an accumulation of amino acid substitutions over time. The appearance of substitutions in the envelope genes was associated with increased antienvelope antibody titers. However, extensive mutations were not incorporated into hypervariable region 1 (HVR1). A comparison of the nonsynonymous substitution rate/synonymous substitution rate was made at various time points to analyze selective pressure. The highest level of selective pressure occurred during the acute phase and decreased as the infection continued. The nonsynonymous substitution rate was initially higher than the synonymous substitution rate but decreased over time from 3.3 x 10(-3) (chimpanzee 1535) and 3.2 x 10(-3) (chimpanzee 1536) substitutions/site/year at week 26 to 1.4 x 10(-3) (chimpanzee 1535) and 1.7 x 10(-3) (chimpanzee 1536) at week 216, while the synonymous substitution rate remained steady at approximately 1 x 10(-3) substitutions/site/year. Analysis of PCR products using single-stranded conformational polymorphism indicated a low level of heterogeneity in the viral genome. The results of these studies confirm that the persistence of infection is not solely due to changes in HVR1 or heterogeneity and that the majority of variants observed in natural infections could not arise simply through mutation during the time period most humans and chimpanzees are observed. These data also indicate that immune pressure and selection continue throughout the chronic phase.

Topics: Amino Acid Substitution; Animals; Ape Diseases; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Pan troglodytes; Polymorphism, Single-Stranded Conformational; Selection, Genetic; Viral Envelope Proteins; Viral Proteins

Most RNA viruses have evolved mechanisms to avoid neutralizing antibody responses, and it is generally believed that variability of envelope-encoding regions is the major molecular basis of this phenomenon. However, it has been hypothesized that other mechanisms can be involved. Recent experimental data indicate that in hepatitis C virus (HCV) infection, the anti-envelope humoral response includes cross-reactive antibody clones able to neutralize vesicular stomatitis virus (VSV) pseudotypes containing HCV E1 and E2 glycoproteins (HCV/VSV pseudotype) as well as other clones devoid of such activity. In this work, we demonstrate that natural infection with a large variety of HCV isolates belonging to different genotypes elicits HCV/VSV pseudotype-neutralizing cross-reactive anti-envelope antibodies together with clones unable to neutralize this pseudovirus. This was shown by designing a novel strategy for quantitation of serum antibodies binding selectively to single viral cross-reactive conformational epitopes. These data can be useful not only for a better understanding of the virus-host interplay in important viral diseases, but also for the development of an effective anti-HCV vaccine.

Topics: Antibodies, Monoclonal; Cross Reactions; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoglobulin Fab Fragments; Neutralization Tests; Recombinant Proteins; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

The N-terminal end of the hepatitis C virus (HCV) envelope glycoprotein E2 contains a stretch of 27 amino acids that exhibit increased variability. This hypervariable region 1 (HVR-1), as it is normally referred to, is thought to contain epitopes that come under humoral immune attack. In the present study, 10 patients (5 children and 5 adults) with humoral immune defects and chronic HCV infection were investigated, to see how HVR-1 sequences behave over time in these patients who are unable to produce antibodies. Amplicons of this region showed little or no variation at all over time, indicating that quasispecies variation in this region is driven by the host's humoral immune response.

Topics: Adolescent; Adult; Agammaglobulinemia; Amino Acid Sequence; Child; Common Variable Immunodeficiency; Complementarity Determining Regions; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Male; Molecular Sequence Data; Sequence Analysis, DNA; Viral Envelope Proteins

A simple and reproducible hepatic immunohistochemical staining (IHS) for hepatitis C virus (HCV) is not available. We aimed to validate hepatic IHS with monoclonal antibody (Mab) IG222, directed against the HCV-envelope 2 (E2) protein.. A three-step indirect immunoperoxidase method was used for frozen sections and a two-step indirect EnVision technique was used for paraffin-embedded sections.. Naturally or in vitro HCV infected primary human hepatocytes were immunoreactive to HCV-E2. In the patient study (n=253), IHS had a sensitivity of 96% and a specificity of 91%. Six patients who showed positivity in the liver with Mab IG222, but remained anti-HCV and HCV-RNA negative, had hepatitis C-like changes in their liver biopsy. In one patient HCV-RNA could be detected in the liver biopsy. We confirmed early graft reinfection in patients transplanted for HCV-related disease (34 patients with serial biopsies). Treatment for acute cellular rejection with steroids was associated with an increase in staining intensity. In nine patients with clearance of HCV-RNA during antiviral therapy, seven achieved negativation of immunoreactivity and two a marked reduction.. The IHS with Mab IG222 is an accurate tool for diagnosis and clinical management of chronic hepatitis C.

Topics: Animals; Antibodies, Monoclonal; Antiviral Agents; Cells, Cultured; Cross-Sectional Studies; Hepatitis C Antibodies; Hepatitis C, Chronic; Hepatocytes; Immunohistochemistry; Liver; Liver Transplantation; Longitudinal Studies; Mice; Mice, Inbred BALB C; Recurrence; Viral Envelope Proteins

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of non-aggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

Topics: Adolescent; Adult; Aged; Animals; Cell Line; Female; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Immunoglobulin G; Male; Mice; Mice, Inbred BALB C; Middle Aged; Protein Engineering; Recombinant Proteins; Simplexvirus; Transfection; Vaccination; Viral Envelope Proteins

Nonstructural 5A (NS5A) and the second envelope (E2) proteins of hepatitis C virus (HCV) have the potential to block interferon (IFN)-induced RNA-dependent protein kinase (PKR) and may therefore interfere with the response to IFN therapy, but controversy still exists regarding the relevance of this. This study aimed to assess whether mutations in these regions correlated with the response to combination therapy, IFN and ribavirin. Pretreatment parameters were analysed in 57 HCV-1b patients who had received IFN-alpha2b (3 or 5 MU three times weekly) and ribavirin (800-1200 mg per day) for 24 weeks. The amino acid sequences of the NS5A and PKR-eIF2alpha phosphorylation homology domain (E2-PePHD) were deduced from the corresponding coding sequence, which were determinated by direct sequencing of the HCV genome amplified by the polymerase chain reaction. Twenty (36%) patients achieved a sustained virological response (SVR). The mean number of amino acid substitutions in the NS5A-PKR binding domain (2209-2274), interferon sensitivity-determining region (ISDR) (2209-2248), and E2-PePHD sequence (659-670) in patients with and without SVR were 4.53 +/- 3.31 vs 2.83 +/- 1.78 (P = 0.094), 2.45 +/- 2.74 vs 1.03 +/- 1.32 (P = 0.042) and 0.25 +/- 0.70 vs 0.03 +/- 0.17 (P = 0.109), respectively. Patients with a mutant-type (>/= 4) NS5A-ISDR had a higher rate of SVR (six of nine, 67%) than those with wild-type (five of 22, 23%) (P = 0.038). Stepwise multiple logistic regression analysis of the factors (age, gender, viral load, cirrhosis rate, IFN dosage and amino acid substitutions) revealed that the mutation in NS5A-ISDR (>/= 4 vs < 4) was the only independent variable of treatment outcome. Our study showed that NS5A-ISDR mutations were correlated with the SVR to combination therapy in chronic HCV-1b patients in Taiwan.

Topics: Amino Acid Sequence; Antiviral Agents; Base Sequence; Drug Therapy, Combination; Eukaryotic Initiation Factor-2; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Point Mutation; Recombinant Proteins; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; RNA, Viral; Sequence Alignment; Viral Envelope Proteins; Viral Nonstructural Proteins

We herein focused on identifying biological factors possibly involved in non-parenteral transmission of hepatitis C virus (HCV), such as HCV excretion patterns and antibody-based immunity to the virus in saliva and/or cervicovaginal secretions (CVS).. Paired blood, saliva and cervicovaginal lavage samples were obtained from HCV-RNA plasma-positive hemoglobin (Hb) antigen and HIV-seronegative, HCV-seropositive males (n=13) and females (n=21). HCV-specific antibodies were detected by ELISA in paired samples, and HCV-RNA was detected in cell-free and cell-associated body fluids.. Antibodies to E1 HCV surface glycoprotein of the IgG and IgA isotypes showed similar, but less pronounced, profiles as IgG and IgA to E2. HCV-specific IgG and IgA in mucosal fluids likely originated predominantly from the systemic compartment, because HCV-specific mucosal immunoglobulins involved primarily monomeric antibodies, including monomeric IgA, and because their specific activities for HCV antigens in corporeal fluids were similar to those in paired serum (Se). Viral shedding in saliva or CVS was restricted to cell-associated, non-replicating strand((+)) HCV-RNA in 42% (12 out of 28) of saliva and in 19% (four out of 21) of cervicovaginal fluids.. The association in body fluids of HCV-specific IgG, and to a lesser extent IgA, directed to E1/E2 surface glycoproteins (which may block critical steps of virus-cell interactions), of undetectable free viral RNA, and of occasional non-replicating cell-associated HCV, suggests a resulting poor infectivity of saliva or cervicovaginal fluid in chronically HCV-infected individuals. Taken together, these observations provide the basis for the low risk of non-parenteral transmission of HCV infection.

Topics: Adult; Aged; Antibodies, Viral; Antibody Formation; Body Fluids; Cervix Uteri; Female; Hemoglobins; Hepatitis C, Chronic; Humans; Immunoglobulin A; Immunoglobulin G; Male; Middle Aged; Mucous Membrane; Saliva; Serum Albumin; Vagina; Viral Envelope Proteins; Virus Shedding

Superinfection of different viral strains within a single host provides an opportunity for studying host-virus and virus-virus interactions, including viral interference and genetic recombination, which cannot be studied in infections with single viral strains. Hepatitis C virus (HCV) is a positive single-strand RNA virus that establishes persistent infection in as many as 85% of infected individuals. However, there are few reports regarding coinfection or superinfection of HCV. Because of the lack of tissue culture systems and small animal models supporting efficient HCV replication, we explored these issues in the setting of liver transplantation where both recipient and donor were infected with different HCV strains and therefore represent a distinct model for HCV superinfection. Serial serum samples collected at multiple time points were obtained from 6 HCV-positive liver donor/recipient pairs from the National Institute of Diabetes and Digestive and Kidney Diseases liver transplantation database. At each time point, HCV genotype was determined by both restriction fragment length polymorphism analysis and phylogenetic analysis. Furthermore, we selectively sequenced 3 full-length HCV isolates at the earliest time points after liver transplantation, including both 5' and 3' ends. Detailed genetic analyses showed that only one strain of HCV could be identified at each time point in all 6 cases. Recipient HCV strains took over in 3 cases, whereas donor HCV strains dominated after liver transplantation in the remaining 3 cases. In conclusion, in all 6 cases studied, there was no genetic recombination detected among HCV quasispecies or between donor and recipient HCV strains.

Topics: Adult; Amino Acid Substitution; Cloning, Molecular; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Transplantation; Male; Middle Aged; Recombination, Genetic; RNA, Viral; Superinfection; Tissue Donors; Transplantation; Viral Envelope Proteins

Combination therapy with interferon (IFN) and ribavirin is the current standard treatment for chronic hepatitis C, but the efficacy is still not satisfactory, especially for genotype 1b. NS5A and E2 proteins of hepatitis C virus (HCV) may repress the IFN-induced RNA-dependent protein kinase (PKR), and thus have the potential to influence the response of HCV to IFN therapy; however, this issue remains controversial.. Nucleotide sequences of the PKR-eIF2alpha phosphorylation homology domain (E2-PePHD) and PKR-binding domain (NS5A-PKR bd) of the HCV genome were analyzed by amplification and direct sequencing in 30 HCV genotype 1b patients who had been treated with IFN and ribavirin.. Nine (30%) patients achieved a sustained virological response (SVR) to combination therapy. Pretreatment variables and amino acid substitutions were compared between responders and non-responders. The responders were younger than non-responders (37.2 +/- 10.4 vs. 45.4 +/- 9.5 years, P = 0.017), whereas no significant statistical differences were found in the number of amino acid substitutions in NS5A and E2-PePHD regions between the two groups.. Genetic heterogeneity in NS5A and E2-PePHD regions of the HCV genome may not serve as a predictor for treatment outcome with combination therapy in Taiwanese patients with chronic HCV genotype 1b infection.

Topics: Adult; Amino Acid Substitution; Antiviral Agents; Drug Therapy, Combination; eIF-2 Kinase; Female; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon Type I; Interferon-alpha; Male; Middle Aged; Phosphorylation; Point Mutation; Polyethylene Glycols; Polymerase Chain Reaction; Recombinant Proteins; Ribavirin; Sequence Analysis, DNA; Treatment Outcome; Viral Envelope Proteins; Viral Nonstructural Proteins

The association of the severity of liver disease and the molecular evolution of hepatitis C virus (HCV) during chronic infection remains unclear and controversial. To address this we have studied the interpatient variability in the nucleotide sequence of two regions of the HCV genome, E1/E2, which contain the hypervariable region 1 and the nonstructural NS5b region, in a cohort of Irish female patients who were all recipients of a single source of HCV genotype 1b-contaminated anti-D immunoglobulin in 1977 and 1978 and who over the subsequent 20 years developed a spectrum of liver disease. In addition, quasispecies analysis was used to evaluate intrapatient variability in the E1/E2 region in four patients with mild and four with severe disease. Phylogenetic and evolutionary rate analyses of the nucleotide sequences demonstrated that there was no significant difference between those who developed mild disease and those who had progressed to severe disease or cirrhosis. These findings suggest that other factors, either additional viral or host, may be important in the pathogenesis and clinical outcome of chronic hepatitis C virus infection.

Topics: Amino Acid Sequence; Cohort Studies; Evolution, Molecular; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Liver Cirrhosis; Molecular Sequence Data; Nucleotides; Phylogeny; Viral Envelope Proteins; Viral Nonstructural Proteins

To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.. The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody.. The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.. We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.

Topics: Amino Acid Sequence; Antibody Specificity; Bacteriophages; Base Sequence; Escherichia coli; Gene Expression; Gene Library; Genetic Testing; Hepatitis C, Chronic; Humans; Immunoglobulin Fragments; Immunohistochemistry; Liver; Molecular Sequence Data; Viral Envelope Proteins

Hepatitis C virus (HCV) is the major causative agent of blood-borne non-A, non-B hepatitis. Although a strong humoral response is detectable within a few weeks of primary infection and during viral persistence, the role played by antibodies against HCV envelope glycoproteins in controlling viral replication is still unclear. We describe how human monoclonal anti-HCV E2 antibody fragments isolated from a chronically HCV-infected patient differ sharply in their abilities to neutralize infection of HepG2 cells by a vesicular stomatitis virus pseudotype bearing HCV envelope glycoproteins. Two clones were able to neutralize the pseudotype virus at a concentration of 10 micro g/ml, while three other clones completely lacked this activity. These data can explain the lack of protection and the possibility of reinfection that occur even in the presence of a strong antiviral antibody response.

Topics: Antibodies, Monoclonal; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoglobulin Fab Fragments; Neutralization Tests; Recombinant Proteins; Tumor Cells, Cultured; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

To investigate the variations of HCV core and E2 region epitopes during transfusion of activated immune cells.. Four patients receiving transfusion of activated immune cells were under continuously observation. HCV titers were measured by quantitive PCR. HCV core and E2 regions were cloned and sequences were analyzed by computer software.. During the follow-up the serum ALT levels and the HCV virus titers fluctuated greatly in each of these persons. After transfusion, no significant variations were observed in HCV core and E2 coding regions.. The alteration of host immune attacks could induce the fluctuations of HCV load without any mutations in the currently observed coding genes of the epitopes.

Topics: Adult; Aged; Alanine Transaminase; Epitopes; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Immunotherapy; Male; Middle Aged; Viral Core Proteins; Viral Envelope Proteins; Viral Load

Hepatitis C virus (HCV) exists as a complex swarm of genetically related viruses known as a quasispecies. Recent work has shown that quasispecies complexity and evolutionary rates are associated with the outcome of acute infection. Knowledge of how the virus population evolves at different stages of chronic infection is less clear. We have studied rates of evolution of the first hypervariable region (HVR1) of the E2 envelope protein in six individuals with disparate liver disease severity. These data show that virus populations present in individuals with mild non-progressive liver disease evolve in a typical Darwinian fashion, with a consistent accumulation of non-synonymous (amino acid-changing) substitutions. By contrast, the virus population remains relatively static in individuals with severe progressive liver disease. Possible mechanisms for this disparity are discussed.

Topics: Alanine Transaminase; Amino Acids; Base Sequence; Binding Sites; Evolution, Molecular; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Liver; Liver Diseases; Molecular Sequence Data; Mutagenesis; Nucleotides; Phylogeny; Protein Sorting Signals; Sequence Analysis, DNA; Viral Envelope Proteins; Viral Load

The aim of this study was to determine whether expression of hepatitis C virus proteins alters hepatic morphology or function in the absence of inflammation.. Transgenic C57BL/6 mice with liver-specific expression of RNA encoding the complete viral polyprotein (FL-N transgene) or viral structural proteins (S-N transgene) were compared with nontransgenic littermates for altered liver morphology and function.. FL-N transcripts were detectable only by reverse-transcription polymerase chain reaction, and S-N transcripts were identified in Northern blots. The abundance of viral proteins was sufficient for detection only in S-N transgenic animals. There was no inflammation in transgenic livers, but mice expressing either transgene developed age-related hepatic steatosis that was more severe in males. Apoptotic or proliferating hepatocytes were not significantly increased. Hepatocellular adenoma or carcinoma developed in older male animals expressing either transgene, but their incidence reached statistical significance only in FL-N animals. Neither was ever observed in age-matched nontransgenic mice.. Constitutive expression of viral proteins leads to common pathologic features of hepatitis C in the absence of specific anti-viral immune responses. Expression of the structural proteins enhances a low background of steatosis in C57BL/6 mice, while additional low level expression of nonstructural proteins increases the risk of cancer.

Topics: Animals; Apoptosis; Cell Division; Fatty Liver; Female; Gene Expression Regulation, Viral; Hepacivirus; Hepatitis C, Chronic; Liver Cirrhosis; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; RNA, Messenger; Transgenes; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Structural Proteins

Despite recent treatment advances, the majority of patients with chronic hepatitis C fail to respond to antiviral therapy. Although the genetic basis for this resistance is unknown, accumulated evidence suggests that changes in the heterogeneous viral population (quasispecies) may be an important determinant of viral persistence and response to therapy. Sequences within hepatitis C virus (HCV) envelope 1 and envelope 2 genes, inclusive of the hypervariable region 1, were analyzed in parallel with the level of viral replication in serial serum samples obtained from 23 patients who exhibited different patterns of response to therapy and from untreated controls. Our study provides evidence that although the viral diversity before treatment does not predict the response to treatment, the early emergence and dominance of a single viral variant distinguishes patients who will have a sustained therapeutic response from those who subsequently will experience a breakthrough or relapse. A dramatic reduction in genetic diversity leading to an increasingly homogeneous viral population was a consistent feature associated with viral clearance in sustained responders and was independent of HCV genotype. The persistence of variants present before treatment in patients who fail to respond or who experience a breakthrough during therapy strongly suggests the preexistence of viral strains with inherent resistance to IFN. Thus, the study of the evolution of the HCV quasispecies provides prognostic information as early as the first 2 weeks after starting therapy and opens perspectives for elucidating the mechanisms of treatment failure in chronic hepatitis C.

Topics: Adult; Antiviral Agents; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Recombinant Proteins; Treatment Outcome; Viral Envelope Proteins

Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins.. Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays.. Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL.. These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.

Topics: Amino Acid Sequence; Antigens, Viral; Cell Line; Conserved Sequence; Epitopes; Hepacivirus; Hepatitis C, Chronic; HLA-A2 Antigen; Humans; In Vitro Techniques; Molecular Sequence Data; Protein Binding; T-Lymphocytes, Cytotoxic; Viral Envelope Proteins

To determine whether the hepatitis C virus (HCV) E2 PePHD sequence (aa 659-670; PKR-eIF2alpha phosphorylation homology domain) is the determinant for the response of interferon treatment, we have analyzed PePHD sequences in HCV-infected patients who had received interferon-alfa treatment. The PePHD sequence from all (6/6) of the patients, who are non- or partial responders to the interferon treatment, is the wild-type sequence (RSELSPLLL-TT, consensus sequence of HCV-1a and HCV-1b). However, there are sequence variations from more than half (5/9) of the patients, who are complete responders to the treatment. We have also analyzed the NS5A ISDR sequence (aa 2209-2248, interferon sensitivity-determining region) variation in HCV-1b-infected patients. No such correlation has been observed. Thus, our data suggest that HCV E2 should play a more important role than NS5A in determining the interferon responses.

Topics: Adult; Aged; Amino Acid Sequence; Drug Resistance, Microbial; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Mutation; Viral Envelope Proteins; Viral Load

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

Topics: Acute Disease; Amino Acid Sequence; Case-Control Studies; Epitopes; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunoenzyme Techniques; Molecular Sequence Data; Sequence Homology, Amino Acid; Viral Envelope Proteins

Two genomic regions of hepatitis C virus (HCV), the interferon sensitivity-determining region (ISDR) of the non-structural 5A gene (NS5A) and the protein kinase-RNA activated (PKR)-eukariotic transcription factor (eIF2-alpha) phosphorylation homology domain (PePHD) of the structural E2 gene, interact in vitro with the interferon-inducible cellular PKR protein kinase. Mutations within these regions might, therefore, influence the response to interferon therapy. Viral load at baseline and sequence heterogeneity of HCV in NS5A and E2 regions was studied in 74 HCV-1b and in 12 HCV-3a infected patients with chronic hepatitis C who were treated with interferon. As previously reported by us, in a smaller series of patients in which the ISDR region was analyzed [Saiz et al. (1998) Journal Infectious Diseases 177:839-847], in the present study a low viral load and a high number of amino acid mutations within the ISDR, but not within the PePHD region, were significantly associated with long-term response to interferon among HCV-1b infected patients. No relationship between these viral features and response to therapy was disclosed in patients infected with HCV-3a.

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Female; Genetic Variation; Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Recombinant Proteins; RNA, Viral; Treatment Outcome; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

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.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antibody Specificity; Cross Reactions; Female; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Immunodominant Epitopes; Molecular Sequence Data; Peptide Library; Rabbits; Viral Envelope Proteins; Viral Hepatitis Vaccines

CD8(+) cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.

Topics: Acute Disease; Amino Acid Sequence; Animals; Antigenic Variation; Cell Line; Epitopes; Follow-Up Studies; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Hepatitis C, Chronic; Histocompatibility Antigens Class I; Molecular Sequence Data; Mutation; Pan troglodytes; Remission, Spontaneous; RNA, Viral; Sequence Alignment; Sequence Homology, Amino Acid; T-Lymphocytes, Cytotoxic; Viral Envelope Proteins; Viral Nonstructural Proteins

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P

Topics: Adult; Amino Acid Sequence; Antiviral Agents; Base Sequence; DNA, Viral; Female; Genetic Heterogeneity; Genome, Viral; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Recombinant Proteins; Retrospective Studies; Sequence Homology, Amino Acid; Viral Envelope Proteins

We have found differences among the populations of hepatitis C virus sequences in serum, peripheral blood mononuclear cells (PBMCs), and various tissues in patients with chronic hepatitis C. These results are compatible with the existence of independent viral compartments in the infected host. Our results also suggest that PBMCs, and probably various tissues, can selectively adsorb viral subpopulations differing in the E2 region.

Topics: 5' Untranslated Regions; Adsorption; Base Sequence; Hepacivirus; Hepatitis C, Chronic; Humans; Leukocytes, Mononuclear; Liver Failure; Molecular Sequence Data; Sequence Homology, Nucleic Acid; Tissue Distribution; Viral Envelope Proteins; Virion; Virus Replication

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

Topics: Acute Disease; Adult; Aged; Antibodies, Viral; Disease Progression; Evolution, Molecular; Female; Genes, Viral; Genetic Variation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Male; Middle Aged; Molecular Sequence Data; Phylogeny; Prospective Studies; Selection, Genetic; Time Factors; Viral Envelope Proteins; Virus Replication

Hepatitis C virus (HCV) variability was analyzed based upon an isolate which had caused the infection of more than 2500 women in 1978/79. Genome consensus sequences of two isolates obtained from the infectious source (HCV-AD78) and from a chronic hepatitis patient 10 years after the acute infection were determined. The entire open reading frame (ORF) exhibited 3.2 x 10(-3) nucleotide substitutions per site per year (deltant). Core (0.7 x 10(-3) deltant) and NS5B (1.9 x 10(-3) deltant) were found to be most conserved genes, while E2 (4.7 x 10(-3) deltant) with hypervariable region 1 (HVR1) (23 x 10(-3) deltant) was the most variable followed by p7 (4.2 x 10(-3) deltant). In the entire ORF transitions were 4.5 times more frequent than transversions while for the HVR1 this bias was turned. As an indicator of relative selective pressure on the proteins the rates of nonsynonymous to synonymous substitutions (dN/dS) were determined. The obtained values exceeded 1.0 only for E2 (dN/dS = 1.3). A subdivision of the entire ORF into 88 overlapping sections, each containing 300 nucleotides, led to a more precise analysis of HCV diversity. Besides for E2 an increased variability was mainly detected for three other regions: (a) the C terminal neighbouring region of E2 including p7, (b) the genome fragment extending from approximately the middle of NS3 to NS4B, and (c) the segment corresponding to the C-terminus of the NS5A protein. The variable region in NS5A was situated carboxyterminal to the predicted interferon sensitivity determining region (ISDR). These results suggest which regions other than HVR1 might contribute to persistence of the virus by the mechanism of immunescape.

Topics: Base Sequence; Consensus Sequence; Disease Outbreaks; DNA, Viral; Female; Genetic Variation; Germany; Hepacivirus; Hepatitis C, Chronic; Humans; Molecular Sequence Data; Open Reading Frames; Time Factors; Viral Core Proteins; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins

In order to study the significance of HCVE2/NS1 relative conservation region antigen.. EIA was established with HCVE2/NS1 relative conservation polypeptide antigen, as well as serum HCV RNA and ALT in patients with HCV infection.. The positive rate of 96 cases patients who were infected by HCV is 41.67%, acute 13.55% and chronic 25.04%, and we found that there were 3 cases weak positive in 40 healths; Agreement rate is 62.5% in 40 cases of the positive of anti-E2/NS1 in sera with HCVRNA, and they have relation. In ALT level in sera, acute infection of HCV is higher than chronic (P < 0.05).. There are virus necleic acid in sera when anti-HCVE2/NS1 appearence positive; however, antibody of anti-E2/NS1 relative conservation region has the some neutralive actions, and this humor immunity may have the actions in clearing virus, but wether anti-body of anti-E2/NS1 relative conservation of HCV is or not neutralive antibody well be investigated further.

Topics: Adult; Alanine Transaminase; Enzyme-Linked Immunosorbent Assay; Female; Hepatitis C; Hepatitis C Antibodies; Hepatitis C Antigens; Hepatitis C, Chronic; Humans; Male; Viral Envelope Proteins

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.

Topics: Adolescent; Adult; Aged; Alanine Transaminase; Antiviral Agents; Evolution, Molecular; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Phylogeny; Recombinant Proteins; RNA, Viral; Viral Envelope Proteins

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 m

Topics: Adult; Antiviral Agents; Drug Therapy, Combination; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Middle Aged; Ribavirin; Treatment Outcome; Viral Envelope Proteins; Viral Load; Viral Nonstructural Proteins

To determine whether there was a correlation between the kinetics or frequency of antibody to mammalian-derived hepatitis C virus (HCV) second envelope protein (E2) and development of chronicity or self-limitation of HCV infections, serial sera were examined for anti-E2, anti-HCV with confirmation with Matrix 2.0 (Abbott Laboratories, Abbott Park, IL), and reverse transcriptase-polymerase chain reaction (RT-PCR) from 6 cases of self-limited infection and 6 cases of chronic infection in chimpanzees, and from 5 cases of self-limited infection and 3 cases of chronic infection in patients. Anti-E2 developed earlier, more frequently, and to higher titer in chimpanzees and patients who were developing chronic infection than in those with self-limited infections. Thus anti-E2 is unlikely to play a role in self-limitation of the infection. However, long-term persistence of anti-E2 correlates with chronic infection. There was little or no correlation between the timing of development of anti-E2 and anti-HCV.

Topics: Animals; Blood Transfusion; Follow-Up Studies; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Pan troglodytes; Prospective Studies; Time Factors; Viral Envelope Proteins

The prevalence of anti-E2 antibody in persons chronically infected with hepatitis C virus (HCV) is high irrespective of viral genotype, and this cross-reactive antibody is thought to react with a conformational epitope. To investigate the characteristics of this anti-E2 antibody, the immunoreactivity of sera from HCV-1b-infected patients was measured against various modified forms of E2 glycoprotein derived from HCV-H (genotype 1a) by an immunofluorescence technique. Twelve of 18 patients were positive for anti-E2 antibody, and 10 of the 12 required a minimal amino acid (aa) region including aa 406-644 for strong reactivity, suggesting that the major E2 antibody has a conformational epitope in this region. Subsequent analysis using mutant E2 glycoproteins designed to lose N-glycosylation potential at varying sites revealed seven important N-glycosylation sites in this region. Four of these (aa 423, 430, 448, and 576) are indispensable for an antibody response.

Topics: Amino Acid Sequence; Cross Reactions; Epitopes; Fluorescent Antibody Technique; Genotype; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Melanoma; Molecular Sequence Data; Promoter Regions, Genetic; Protein Conformation; Recombinant Proteins; Sensitivity and Specificity; Sequence Alignment; Tumor Cells, Cultured; Viral Envelope Proteins

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

Topics: Adolescent; Adult; Aged; Antiviral Agents; Base Sequence; Female; Genetic Variation; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon-alpha; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Sequence Alignment; Sequence Homology, Nucleic Acid; Viral Envelope Proteins

The relatively high variability of the hepatitis C virus (HCV) envelope proteins E1 and E2 suggests that parts of these proteins other than the hypervariable region 1 (HVR1) might be involved in the induction of virus neutralizing antibodies. To test this hypothesis, two HCV proteins, pE1 and pE2 delta, were generated by in vitro translation. They represent amino acids 174-337 of E1 and 411-688 of E2, respectively, of isolate HCV-AD78; the protein pE2 delta contained no HVR1. As a control, protein pG.HVR1, which represents amino acids 384-410 of HVR1 of isolate HCV-AD78, was expressed separately. These three proteins were used in an immunoprecipitation assay to detect the presence of antiviral antibodies in sera of patients infected with the same isolate of HCV (HCV-AD78). Sera were obtained 4-8 months postinfection from patients who later resolved an acute infection or developed chronic liver disease. A high prevalence of antibodies (up to 85.7%) against pE1 and pE2 delta could be detected in both groups of patients, suggesting that these forms of the HCV envelope proteins contain B-cell epitopes. The antibody responses against proteins pE1 and pE2 delta did not differ significantly between patients with resolving or chronic infection, whereas antibodies against protein pG.HVR1 were associated with resolution of infection. Rabbit antisera raised against pE1 and pE2 delta were tested for their ability to neutralize the binding of HCV to susceptible cells in tissue cultures. The results suggested that although a few B-cell epitopes outside of HVR1 can induce virus neutralizing antibodies, these antibodies are probably not associated with the resolution of infection.

Topics: Animals; Cell Line; Epitopes, B-Lymphocyte; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Rabbits; Viral Envelope Proteins

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.

Topics: Amino Acid Sequence; Animals; Antigenic Variation; Bacteriophage M13; Cloning, Molecular; Cross Reactions; Epitopes, B-Lymphocyte; Female; Gene Library; Genetic Variation; Genetic Vectors; Hepacivirus; Hepatitis C Antibodies; Hepatitis C, Chronic; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Structure-Activity Relationship; Viral Envelope Proteins

The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus (ALVAC) expressing HCV-capsid/E1/E2/NS2/NS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1/2663 and 1/27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV-CTL response and virological or histological status.

Topics: Animals; Avipoxvirus; COS Cells; Hepacivirus; Hepatitis C Antigens; Hepatitis C, Chronic; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Pan troglodytes; Sensitivity and Specificity; T-Lymphocytes, Cytotoxic; Viral Envelope Proteins; Viral Nonstructural Proteins