Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world''s population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4⁺ and CD8⁺ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV⁺ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8⁻ Jurkat cells and CD4⁺ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.     

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3<Superscript>+</Superscript>, CD14<Superscript>+</Superscript> and CD19<Superscript>+</Superscript>

Background

Although hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3⁺, CD14⁺ and CD19⁺. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.

Methods

PBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3⁺, CD14⁺, CD19⁺ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.

Results

Negative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3⁺ cells then in 8/26 (30.8%) of CD14⁺ and CD19⁺ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3⁺ (2.4%), then in CD19⁺ (1.2%), and much lower in CD14⁺ (0.4%) cells.

Conclusions

Our results indicate that CD3⁺ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

     

Anti-Hepatitis C Virus T-Cell Immunity in the Context of Multiple Exposures to the Virus

Characterisation of Hepatitis C virus (HCV)-specific CD8⁺ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8⁺ T-cell targets. Peptides representing these putative HCV CD8⁺ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design.     

Characterization of the specific CD4+ T cell response against the F protein during chronic hepatitis C virus infection

Background

The hepatitis C virus (HCV) Alternate Reading Frame Protein (ARFP or F protein) presents a double-frame shift product of the HCV core gene. We and others have previously reported that the specific antibodies against the F protein could be raised in the sera of HCV chronically infected patients. However, the specific CD4⁺ T cell responses against the F protein during HCV infection and the pathological implications remained unclear. In the current study, we screened the MHC class II-presenting epitopes of the F protein through HLA-transgenic mouse models and eventually validated the specific CD4⁺ T cell responses in HCV chronically infected patients.

Methodology

DNA vaccination in HLA-DR1 and-DP4 transgenic mouse models, proliferation assay to test the F protein specific T cell response, genotyping of Chronic HCV patients and testing the F-peptide stimulated T cell response in the peripheral blood mononuclear cell (PBMC) by in vitro expansion and interferon (IFN)- γ intracellular staining.

Principal Findings

At least three peptides within HCV F protein were identified as HLA-DR or HLA-DP4 presenting epitopes by the proliferation assays in mouse models. Further study with human PBMCs evidenced the specific CD4⁺ T cell responses against HCV F protein as well in patients chronically infected with HCV.

Conclusion

The current study provided the evidence for the first time that HCV F protein could elicit specific CD4⁺ T cell response, which may provide an insight into the immunopathogenesis during HCV chronic infection.     

Discovery of velpatasvir (GS-5816): A potent pan-genotypic HCV NS5A inhibitor in the single-tablet regimens Vosevi® and Epclusa®

Direct-acting antiviral inhibitors have revolutionized the treatment of hepatitis C virus (HCV) infected patients. Herein is described the discovery of velpatasvir (VEL, GS-5816), a potent pan-genotypic HCV NS5A inhibitor that is a component of the only approved pan-genotypic single-tablet regimens (STRs) for the cure of HCV infection. VEL combined with sofosbuvir (SOF) is Epclusa^®, an STR with 98% cure-rates for genotype 1–6 HCV infected patients. Addition of the pan-genotypic HCV NS3/4A protease inhibitor voxilaprevir to SOF/VEL is the STR Vosevi^®, which affords 97% cure-rates for genotype 1–6 HCV patients who have previously failed another treatment regimen.     

Chronic hepatitis C viral infection reduces NK cell frequency and suppresses cytokine secretion: Reversion by anti-viral treatment

Impaired activity of NK (natural killer) cells has been proposed as a mechanism contributing to viral persistence and chronic infection in hepatitis C (HCV) infection. We aimed to assess the impact of HCV infection on NK cells regarding frequency, subset distribution, and cytotoxic and cytokine secretion functions, as well as IFN-α and ribavirin therapeutic effects on NK cells. Significant reduction of total NK frequency and the CD56^dim16⁺ subset was observed in chronic HCV patients. IFN-γ expression upon stimulation with K562 was severely suppressed but cytotoxicity measured by CD107a expression was maintained. These adverse effects were reversed after treatment with pegylated IFN-α and ribavirin; however, these skewed functions were not recovered in treatment-resistant patients. Thus, HCV chronic infection severely affects NK functions, except for cytotoxicity. Altered NK cell frequency and cytokine secretion by HCV infection may contribute to impaired cellular immune response and virus persistence.     

Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQβ1

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQβ1Leu26 (p value_Meta = 1.24 × 10^?14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQβ1Pro55 (p value_Meta = 8.23 × 10^?11) located in the peptide binding region was positively associated, independently of HLA-DQβ1Leu26. These two amino acids are not in linkage disequilibrium (r² < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1¹03:01, and HLA-DRB1¹01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQβ1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC₅₀ nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10^?38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQβ1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.     

Performance of the automated COBAS AMPLICOR™ system for the detection of hepatitis C virus RNA

Background: The COBAS AMPLICOR^™ (CA) instrument for the amplification and detection steps of the AMPLICOR^™ molecular diagnostic assays has recently been introduced. The system contains a single thermal cycler with two independently controlled heating/cooling blocks, a pipettor, a magnetic particle washer, a photometer and an incubator.Objective: The performance of the CA instrument was evaluated in a routine diagnostic laboratory for the detection of hepatitis C virus (HCV) RNA. The new system was compared with the corresponding microwell plate assay (AMPLICOR HCV Test).Study design: Routine clinical sera (350) from hemodialysis patients and patients with chronic HCV infection and interferon therapy were studied. If discrepant results were obtained, both assays were repeated (specimen preparation, amplification and detection); in addition, the HCV copy number was determined with the AMPLICOR HCV MONITOR^™ Test.Results: There was a correlation between the CA HCV Test and the AMPLICOR HCV Test in 341 of 350 specimens (97%). After resolution of 9 discrepant results, the CA HCV Test gave a sensitivity of 97.8% and a specificity of 99.4%. The most common reason for discrepant results was a low HCV RNA copy number.Conclusion: The CA system was found to be a labor-saving, fast and reliable instrument for the amplification and detection steps of a RT-PCR molecular assay for detection of HCV RNA.     

Hepatitis C virus RNA replication in human stellate cells regulates gene expression of extracellular matrix-related molecules

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, including chronic hepatitis, fibrosis, and cirrhosis. Fibrosis often develops in HCV-infected livers and ultimately leads to cirrhosis and carcinoma. During fibrosis, hepatic stellate cells (HSC) play important roles in the control of extracellular matrix synthesis and degradation in fibrotic livers. In this study, we established a subgenomic replicon (SGR) cell line with human hepatic stellate cells to investigate the effect of HCV RNA replication on HSC. Isolated SGR clones contained HCV RNA copy numbers ranging from 10⁴ to 10⁷ per μg total RNA, and long-term culture of low-copy number SGR clones resulted in markedly increased HCV RNA copy numbers. Furthermore, HCV RNA replication affected gene expression of extracellular matrix-related molecules in both hepatic stellate cells and hepatic cells, suggesting that HCV RNA replication and/or HCV proteins directly contribute to liver fibrosis. The HCV RNA-replicating hepatic stellate cell line isolated in this study will be useful for investigating hepatic stellate cell functions and HCV replication machinery.     

Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+

Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD⁺ ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD⁺. Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting β-oxidation with β-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Conclusion: Therefore, lipid metabolism and alteration of cellular NADH/NAD⁺ ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.     

Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQβ1

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQβ1Leu26 (p value_Meta = 1.24 × 10⁻¹⁴) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQβ1Pro55 (p value_Meta = 8.23 × 10⁻¹¹) located in the peptide binding region was positively associated, independently of HLA-DQβ1Leu26. These two amino acids are not in linkage disequilibrium (r² < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1^∗03:01, and HLA-DRB1^∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQβ1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC₅₀ nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10⁻³⁸). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQβ1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.     

Assessment of immunological changes in Epstein-Barr virus co-infection in Egyptian chronic HCV patients

Epstein-Barr virus (EBV) plays a major role in liver pathology. Similar to other members of the herpesvirus family, EBV establishes a persistent infection in more than 90% of adults. The aim of this study was to evaluate the impact of EBV and chronic hepatitis C co-infection (HCV) on biochemical and immunological responses in patients. The study was conducted in 62 patients and 33 apparently healthy controls. Patients were divided into three groups: group I, consisting of 31 patients with chronic hepatitis C infection (CHC), group II, consisting of eight patients with EBV infection and without HCV infection and group III, consisting of 23 patients with EBV and chronic HCV. The percentage of CD3⁺ cells, helper CD4⁺ cells and CD19⁺ B-cells was measured by flow cytometry. Human interferon-γ (IFN-γ) and interleukin (IL)-15 levels were measured by an ELISA. The levels of liver alanine aminotransferase and aspartate aminotransferase enzymes were higher in EBV/HCV patients compared to that in EBV and HCV mono-infected patients. EBV/HCV patients had significantly reduced percentages of CD3⁺ and CD4⁺ cells compared to EBV patients. Serum IFN-γ levels were significantly reduced in EBV/HCV patients (3.86 pg/mL) compared to CHC patients (6.76 pg/mL) and normal controls (4.69 pg/mL). A significant increase in serum IL-15 levels was observed in EBV/HCV patients (67.7 pg/mL) compared to EBV patients (29.3 pg/mL). Taken together, these observations suggest that HCV and EBV co-infection can potentiate immune response dampening in patients.     

Synthesis and inhibitory activity on hepatitis C virus RNA replication of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analogs

Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replication in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analog 1a as a novel HCV inhibitor. Structural modifications of 1a provided a series of sulfonamides 7 with much more potent HCV RNA replication-inhibitory activity than ribavirin. Compound 7a showed an additive anti-HCV effect in combination with standard anti-HCV therapy (IFN-α plus ribavirin). Since 7a generated reactive oxygen species (ROS) in the ORL8 system and its anti-HCV activity was blocked by vitamin E, its anti-HCV activity may be mediated at least in part by ROS.     

Two methods of heterokaryon formation to discover HCV restriction factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers^1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle ^6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion.To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells ⁹) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection ¹⁰ . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV¹¹) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.     

A new epitope peptide derived from hepatitis C virus 1b possessing the capacity to induce cytotoxic T-lymphocytes in HCV1b-infected patients with HLA-A11, -A31, and -A33

Background Hepatitis C virus (HCV) frequently causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma after long-term persistent infection. Among various genotypes of HCV, HCV1b is resistant to standard interferon therapy, and thus the development of new treatment modality is needed. Results To provide a scientific basis for specific immunotherapy for HCV1b, we investigated HCV1b-derived epitope peptides recognized by human leukocyte antigen (HLA)-A11, -A31, or -A33-restricted cytotoxic T-lymphocytes (CTLs), and report here three novel vaccine candidate peptides selected by both antibody screening and CTL-inducing capacity from among 46 peptides of conserved regions of HCV1b sequences with binding motifs to HLA-A11, -A31, and -A33. Significant levels of IgG reactive to each of the three peptides were detected in the plasma of more than 50% of the HCV1b⁺ patients. One peptide at positions 30–39 of the core protein induced peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A11⁺, -A31⁺, and -A33⁺ patients. The other two peptides at positions 35–43 of the core protein and at positions 918–926 of the non-structural protein 2 also induced peptide-specific CTLs from the PBMCs of HLA-A11⁺ and -A33⁺ patients. Conclusion Therefore, the peptide at positions 30–39 of the core protein could be an appropriate target molecule of specific immunotherapy for all HLA-A11⁺, -A31⁺, and -A33⁺ patients with HCV1b-related diseases.     

Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Broad, multispecific CD4⁺ and CD8⁺ T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8⁺ T-cell responses but low CD4⁺ T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4⁺ T helper responses but no CD8⁺ T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4⁺ T helper responses but no CD8⁺ T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4⁺ and CD8⁺ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.     

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O^Tg) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O^Tg mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.     

The Roles of Endoplasmic Reticulum Overload Response Induced by HCV and NS4B Protein in Human Hepatocyte Viability and Virus Replication

Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EOR) can be activated, which involves the release of Ca²⁺ from ER, production of reactive oxygen species (ROS) and activation of nuclear factor κB (NF-κB). We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca²⁺-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca²⁺-ROS pathway. Moreover, we found that HCV-activated EOR-Ca²⁺-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca²⁺-ROS pathway, whereas activation of EOR-Ca²⁺-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca²⁺-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca²⁺-ROS-NF-κB pathway in natural HCV replication and pathogenesis.