Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection

Spontaneous resolution of hepatitis C virus (HCV) infection is a relatively infrequent event, and these individuals provide a unique opportunity to characterize correlates of protective immunity as an important first step in the development of vaccine candidates. The aim of this study was to directly and comprehensively enumerate HCV-nonstructural protein 3 (NS3) specific CD4(+) and CD8(+) T cells ex vivo from HLA diverse individuals who had been successful in spontaneously resolving HCV infection. We measured interferon gamma (IFN-gamma) production with an ELISPOT assay using magnetic bead-separated CD4(+) or CD8(+) T cells in response to autologous DCs that had been pulsed with 15mer per peptides overlapping by 11 amino acids and spanning all of the NS3 protein (150 total peptides). All subjects with spontaneously recovered HCV infection demonstrated vigorous and multispecific CD4(+) T-cell responses to NS3 peptides, and 6 of 10 subjects demonstrated CD8(+) T-cell responses. More importantly, we identified novel, previously unpredicted antigenic regions, which in most cases elicited high frequencies within a given individual. In conclusion, subjects who have spontaneously eradicated HCV infection up to 35 years earlier demonstrate persistent CD4(+) and CD8(+) T-cell responses specific to NS3. By providing a comprehensive screening of all potential T-cell epitopes contained in the NS3 region, our strategy defines the breadth of the T-cell response and identifies novel, unpredicted specificities.     

Intrahepatic and circulating HLA class II-restricted, hepatitis C virus-specific T cells: functional characterization in patients with chronic hepatitis C

To compare the functional features of circulating and intrahepatic hepatitis C virus (HCV)-specific CD4+ T cells in chronic HCV infection, peripheral blood and liver-infiltrating lymphocytes from 29 patients with chronic hepatitis C were stimulated with structural and nonstructural HCV proteins to produce antigen-specific T-cell lines and clones. Antigen specificity, fine specificity, phenotype, cytokine production, and T-cell receptor (TCR)-vbeta chain expression were analyzed. The results indicate a hierarchy of stimulatory capacity by the different HCV proteins, core being the antigen most frequently recognized by CD4+ intrahepatic lymphocytes, followed by NS4 and NS5. The CD4 response was directed simultaneously against different HCV proteins in individual patients, but fine-specificity analysis indicated that the response was generally focused on a limited number of immunodominant epitopes. Although the narrowly focused nature of this response may favor the emergence of escape mutations, this event was not observed by following-up over time the sequence of 2 epitopes strongly immunodominant for intrahepatic CD4 cells of a patient with chronic HCV infection. In conclusion, simultaneous analysis of peripheral blood and intrahepatic CD4 cells in the same patients indicated a predominant Th1 profile of HCV-specific CD4 cells and suggests a specific compartmentalization of virus-specific T cells into the liver.     

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein:An in silico approach

Objective:To predict immunogenic promiscuous T-cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools.To date,no epitope data are available for the Zika virus in the IEDB database.Methods:We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks.A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using Propred1 and Propred immunoinformatic algorithms respectively.The antigencity predicted score was also calculated for each predicted epitope using the Vaxi Jen 2.0 tool.Results:By using Pro Pred1,23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus.The greatest number of MHC class I binding epitopes were projected within the NS5(21%),followed by Envelope(17%).For MHC class II,greatest number of predicted epitopes were in NS5(19%) followed by the Envelope,NS1 and NS2(17% each).A variety of epitopes with good binding affinity,promiscuity and antigenicity were predicted for both the HLA classes.Conclusion:The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates,which will be able to induce a broad range of immune responses in a heterogeneous HLA population.However,our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.     

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies

Summary. Hepatitis C virus (HCV) is a major health problem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.     

Liver-infiltrating and circulating CD4+ T cells in chronic hepatitis C: immunodominant epitopes,HLA-restriction and functional significance

Abstract: The aim was to assess the specificity and functional significance of liver-infiltrating and peripheral blood T cells in chronic hepatitis C. Peripheral blood mononuclear cells hepatitis C virus from 50 of 58 (86.2%) patients with chronic hepatitis C virus infection and 6 of 28 (21.4%) controls showed a proliferative T cell response to at least one of 16 synthetic peptides covering highly conserved regions of the core, envelope (El) and non-structural regions (NS4) of hepatitis C virus. However, six immunodominant peptides were exclusively recognized by the proliferating blood mononuclear cells from 46 patients with chronic hepatitis C virus infection (79.3%). Fine specificity and HLA-restriction were studied with 15 peptide-specific CD4+ T cell lines and 23 T cell clones isolated from liver tissue and peripheral blood of 12 patients with chronic hepatitis C. It was demonstrated that the peptide-specific response of CD4+ T cells was restricted to the presence of autologous accessory cells and HLA-DR and -DP molecules. Eight peptide-specific T cell lines and five T cell clones derived from liver tissue and peripheral blood, released interferon-γ (200–6600 pg/ml) and tumor necrosis factor-α (100–400 pg/ml) and no or little interleukin-4 (<140 pg/ml) after peptide-specific or mitogeneic stimulation, thus resembling a Th1-like cytokine profile. Patients with active liver disease showed significantly higher proliferative responses to hepatitis C virus core peptides than asymptomatic hepatitis C virus carriers or complete responders to interferon therapy. In conclusion, class II-restricted CD4+ T cell responses to some immunodominant epitopes within the hepatitis core region correlated with disease activity in chronic hepatitis C virus infection. Functionally, liver-infiltrating and peripheral blood T cells released Th1-like cytokines in response to the specific stimulus. Thus, it can be suggested that CD4+ T cells can mediate the pathogenesis of chronic hepatitis C virus induced liver disease.     

Frequencies of HCV-specific effector CD4+ T cells by flow cytometry: correlation with clinical disease stages.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, affecting approximately 2% of the world's population. The immune mechanisms responsible for the highly variable natural history in a given individual are unknown. We used a multiparameter flow cytometric technique to functionally and phenotypically characterize HCV-specific effector T cells in the peripheral blood of 32 individuals with different stages of hepatitis C disease (resolved, mild chronic, advanced chronic) and normal controls. We found the highest frequencies of virus-specific effector cells with an activated memory phenotype (CD45RO+CD69+) in subjects who had resolved HCV infection, either spontaneously or with antiviral therapy. Effector cells from patients with resolved infection produced Th1 type cytokines following stimulation with nonstructural antigens (NS3 and NS4), whereas effector cells from chronically infected patients produced Th1 type cytokines predominantly following stimulation with the HCV core antigen. Stimulation with superantigen staphylococcal enterotoxin (SEB) induced the same levels of cytokine production in the different patient groups. Among the HCV-seropositive patients, viral load inversely correlated with the Th1 effector cell response to NS3. Interleukin (IL)-4 was produced only in response to the control antigens, but not in response to the HCV recombinant proteins. Taken together, these findings suggest that a vigorous HCV-specific CD4+ Th1 response, particularly against the nonstructural proteins of the virus, may be associated with viral clearance and protection from disease progression. Prospective studies using this new flow cytometric assay will be required to determine whether antiviral therapy modifies the frequency, specificity, and function of these virus-specific effector cells.     

Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response

This study was performed to examine the immunologic basis for the apparent ethnic difference in clinical outcome of hepatitis C virus (HCV) infection between African Americans (AA) and Caucasian Americans (CA). To this end, we recruited 99 chronically HCV-infected and 31 spontaneously HCV-cleared subjects for clinical, virologic, and immunologic analysis. In particular, CD4-proliferative T-cell response to genotype 1-derived HCV antigens (core, NS3-NS5) was examined in 82 patients chronically infected with genotype 1 (54 AA, 28 CA) and in all HCV-cleared subjects (14 AA, 17 CA). HCV-specific Th1 response also was examined in 52 chronic and 13 recovered subjects. Our results showed that HCV clearance was associated with a vigorous HCV-specific Th1 response irrespective of ethnic origin. Although the HCV-specific CD4 T-cell response clearly was weaker during chronic infection, AA ethnicity in this setting was associated with a significantly greater CD4-proliferative T-cell response to HCV, particularly to the nonstructural antigens (22% AA vs. 0% CA, P =.007) as well as better clinical parameters of liver disease. Interestingly, most HCV-specific CD4 T-cell proliferative responses in AA patients were unaccompanied by concurrent interferon gamma (IFN-gamma) production, suggesting a dysregulated virus-specific, CD4 T-cell effector function during chronic HCV infection. In conclusion, our results suggest that host ethnicity does influence the clinical outcome and antiviral T-cell response during HCV infection. AA ethnicity is associated with a more robust antiviral CD4 T-cell response than CA ethnicity, although these T cells are limited in direct virus or disease control due to their dysfunctional nature.     

Detection of type 2-like T-helper cells in hepatitis C virus infection: Implications for hepatitis C virus chronicity

One striking clinical feature of hepatitis C virus (HCV) infection is that more than 50% of patients with acute hepatitis C will develop chronic infection. To investigate its possible mechanisms, we examined the activation of type 2-like T-helper (Th2-like) cells relating to the development of chronicity. Peripheral blood CD4⁺ T-cell proliferation and cytokine secretion in response to a panel of recombinant HCV antigens including core (C22), envelope 1 (E1), E2, nonstructural (NS) protein 4 (C100), fusion protein of NS3 and NS4 (C200), and NS5 were assayed in 17 patients with acute hepatitis C. All six patients with self-limited disease had a significant CD4⁺ T-cell proliferation to C22, E1, C100, C200, and NS5, running parallel with the antigen-stimulated secretion of interleukin (IL)-2 and interferon γ (IFN-γ), but not with interleukin (IL)-4 and IL-10, indicating predominant Th1 responses. Among the remaining 11 patients who developed chronicity, 6, 2, and 9 cases showed a specific CD4⁺ T-cell response to C22, C100, and C200, respectively, and the responses were significantly lower than those of cases with recovery in terms of stimulation index (SI) (P < .05) and of antigen-stimulated IL-2 and IFN-γ production. Importantly, IL-4 and IL-10 (Th2 responses) were detectable, and C22-specific Th2-like T-cell clones could be generated from patients with chronicity. The data suggested that activation of Th2 responses in acute hepatitis C patients may play a role in the development of chronicity.(Hepatology 1997 Feb;25(2):449-58)     

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.     

Relation between viral fitness and immune escape within the hepatitis C virus protease

BACKGROUND: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. AIMS: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073-1081 of the NS3 protease was limited by viral fitness. PATIENTS: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. METHODS: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. RESULTS: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073-1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073-1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. CONCLUSIONS: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.     

Hepatitis C virus-specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C

BACKGROUND & AIMS: The role of virus-specific T-helper lymphocyte reactivity in determining the therapeutic response in chronic hepatitis C virus (HCV) infection is not fully understood. METHODS: We studied CD4(+) T lymphocyte proliferation together with interferon (IFN)-gamma and interleukin (IL)-10 production from peripheral blood mononuclear cells in response to 4 HCV antigens (core, NS3, NS4, and NS5) in 25 patients with chronic hepatitis C undergoing antiviral therapy with IFN alone or in combination with ribavirin, prospectively, before, during, and after treatment. RESULTS: HCV-specific T-cell reactivity was uncommon at baseline but increased markedly during antiviral therapy, peaking around treatment weeks 4-8. Resolution of hepatitis C viremia was significantly more likely in patients who developed HCV-specific T-cell proliferation with increased IFN-gamma production. The main difference in T-cell reactivity of patients treated with IFN plus ribavirin was a significantly lower production of IL-10, whereas lymphocyte proliferation was similar to that in patients receiving IFN monotherapy. CONCLUSIONS: Treatment-induced control of hepatitis C viremia is associated with the development of HCV-specific T-cell responses with enhanced IFN-gamma and low IL-10 production. The greater efficacy of combination therapy with IFN-alpha plus ribavirin may be related to its ability to suppress HCV-specific IL-10 production.     

Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence.

The immune response to viral antigens is thought to be responsible for viral clearance and disease pathogenesis during hepatitis C virus (HCV) infection. In chronically infected patients, the T-cell response to the HCV is polyclonal and multispecific, although it is not as strong as the response in acutely infected patients who display a more vigorous T-cell response. Importantly, viral clearance in acutely infected patients is associated with a strong CD4(+) helper T-cell response. Thus, the dominant cause of viral persistence during HCV infection may be the development of a weak antiviral immune response to the viral antigens, with corresponding inability to eradicate infected cells. Alternatively, if clearance of HCV from the liver results from the antiviral effect of T-cell-derived cytokines, as has been demonstrated recently for the hepatitis B virus, chronic HCV infection could occur if HCV is not sensitive to such cytokines or if insufficient quantities of cytokines are produced. Liver cell damage may extend from virally infected to uninfected cells via soluble cytotoxic mediators and recruitment and activation of inflammatory cells forming the necroinflammatory response. Additional factors that could contribute to viral persistence are viral inhibition of antigen processing or presentation, modulation of the response to cytotoxic mediators, immunological tolerance to HCV antigens, mutational inactivation of cytotoxic T lymphocyte (CTL) epitopes, mutational conversion of CTL epitopes into CTL antagonists, and infection of immunologically privileged tissues. Analysis of the basis for viral persistence is hampered because the necessary cell culture system and animal model to study this question do not yet exist.     

Fine specificity of the human T-cell response to the hepatitis B virus preS1 antigen.

The T-cell response to hepatitis B virus envelope antigens was studied in 11 hepatitis B vaccine recipients; 7 were selected to analyze the fine specificity of the T-cell response to the preS1 antigen. Four distinct T-cell epitopes were identified by peripheral blood lymphomononuclear cell stimulation with a panel of short synthetic peptides covering the preS1 sequence. The immunodominance of the preS1 epitopes included within peptides 21-30 and 29-48 was shown by their capacity to restimulate an HLA class II restricted proliferative response of T cells primed with the whole preS1 antigen. Conversely, peptide-specific T cells selected by peripheral blood lymphomononuclear cell stimulation with peptides 21-30 and 29-48 were able to recognize the native preS1 molecule, confirming that these epitopes are actually generated by the intracellular processing of preS1. Finally, amino acid residues essential for T-cell activation by peptide 21-30 were identified using 10 analogues of the stimulatory peptide containing single alanine substitutions. These results may be relevant to the design of efficient synthetic vaccines against hepatitis B virus infection.     

Virus-specific antibody titres in different phases of hepatitis C virus infection

summary . This study aimed to examine anti hepatitis C virus (HCV) antibody titres, their changes and differences in acute, chronic and past HCV infection and to examine them after IFN-α-therapy. Ninety five patients were studied in a cross-sectional investigation and 18 of them were followed long-term. Titres of IgM and IgG antibodies against core, NS3, NS4 (A + B), NS5A proteins were determined by the third generation enzyme immunoassays. Patients with acute hepatitis C developed IgG antibodies against core protein in titres 1/5–1/800 and against individual NS proteins at the same titres. During the first to second month of acute hepatitis C IgG antibody titres to HCV proteins were very low, but they had risen considerably by the fourth to sixth month. Anti-HCV IgM antibodies were found in half the acute hepatitis serum samples, titres were 1/5–1/40. Sixty individuals with chronic hepatitis C showed IgG antibodies against core in titres 1/800–1/40000 and against individual NS proteins in titres 1/5–1/20000. Eight patients with chronic hepatitis C had invariable anti-HCV IgG antibodies over 2–3 years. About 81.7% of chronically infected patients had anti-HCV IgM antibodies in titres 1/5–1/160. Patients with resolution of HCV infection showed only anti-core IgG antibodies (titres 1/5–1/200) or no virus-specific antibodies. Individuals with different response to IFN-α-therapy showed two distinct patterns of anti-HCV antibody titres. Acute and chronic HCV infection may be distinguished by anti-core titres.     

T-cell response relative to genotype and ethnicity during antiviral therapy for chronic hepatitis C

Viral genotype and host ethnicity are important predictors of viral clearance during antiviral therapy for chronic hepatitis C virus (HCV) infection. Based on the role of T cells in natural HCV clearance, we hypothesized that T cells may contribute to the genotypic and ethnic difference in treatment outcome. To test this hypothesis, T-cell response to HCV antigens (core, nonstructural NS3/4 and NS5) and control phytohemagglutinin (PHA) was monitored prospectively and was correlated with virological outcome in 41 patients chronically infected with HCV (27 genotype 1, 14 genotype 2 or 3; 19 black persons, 22 white persons) undergoing combined interferon alfa and ribavirin therapy. Interestingly, in patients with genotype 2 or 3 infection, enhanced virological response coincided with a greater T-cell response to HCV NS3/4 antigen at baseline (50% vs. 15%; P = .026) that augmented further during therapy (29% vs. 4%; P = .035) compared with genotype 1-infected patients. However, HCV-specific T-cell response remained weak in genotype 1-infected patients regardless of virological outcome or ethnicity. Furthermore, virological outcome was associated with a suppressed baseline proliferative response to phytohemagglutinin (P < .03) that increased during therapy (P < .003) independent of ethnicity or genotype. In conclusion, HCV-specific T-cell response was associated with HCV genotype but not with therapeutic clearance of HCV infection. The association between treatment outcome and phytohemagglutinin response suggests more global and antigen-nonspecific mechanisms for therapeutic HCV clearance.     

Hepatitis C viral clearance and antibody reactivity patterns in persons with haemophilia and other congenital bleeding disorders.

We studied hepatitis C virus (HCV) clearance and antibody reactivity patterns in a cohort of 100 haemophiliacs exposed to unsterilized blood products, of whom 25 were antiHCV negative and 75 were antiHCV positive [49 human immunodeficiency virus (HIV) negative and 26 HIV positive]. HCV RNA was measured by the 2.0 bDNA assay and an 'in-house' polymerase chain reaction assay. Antibody reactivity patterns were examined using a recombinant immunoblot assay (RIBA). Prior HCV infection was found in two (8%) of 25 antiHCV negative patients. HCV viraemia persisted in all 26 antiHCV+ patients who were coinfected with HIV. HCV RNA clearance was found in 12 (25%) of 49 antiHCV+, HIV- patients. Viral clearance was associated with younger current age (P < 0.01) and age at infection (P < 0.001), but not with duration of infection or with dose or frequency of clotting factor use. RIBA ratios reflecting an index of each patient's overall reactivity to four HCV epitopes were significantly lower in those with viral clearance (P < 0.0001). Over a period of 15 years, those with viral clearance demonstrated significant loss of reactivity to the NS3, NS4 and NS5 epitopes, while those with viral persistence demonstrated relatively stable reactivities to all epitopes. We conclude that spontaneous HCV RNA clearance in haemophiliacs is age-related and is unlikely to occur in those coinfected with HIV. The loss of antibody reactivity for some epitopes, especially c22 (core), may be a marker for the natural resolution of chronic HCV infection.     

The role of the virus specific T-cell response in acute and chronic HBV and HCV infection

Infections with hepatitis B (HBV) and hepatitis C virus (HCV) are worldwide one of the most frequent causes for chronic liver disease, liver cirrhosis and hepatocellular carcinoma. The mechanisms responsible for the elimination or the persistence of the virus are not well understood. The immunopathogenesis of HBV and HCV infection is primarily mediated by virus specific CD4+- and CD8+-T-cells. During acute infection a strong and multispecific T-cell response against different viral epitopes can be detected and is associated with the clearance of the virus. In case of viral persistence virus specific T-cells contribute to liver inflammation. In this article we summarize the current concepts about the role of the virus specific T-cell response in acute and chronic HBV and HCV infection.     

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection

BACKGROUND/AIMS: It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS: Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS: An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS: Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.     

Quantitative analysis of anti-hepatitis C virus antibody-secreting B cells in patients with chronic hepatitis C

To investigate the quantitative characteristics of humoral immunity in patients with hepatitis C, we established an enzyme-linked immunosorbent spot (ELISpot) assay for detection of anti-hepatitis C virus (HCV)-secreting B cells. Receiver operating characteristic curve analysis demonstrated 100% specificity and 58% to 92% sensitivity for detecting B-cell responses to NS5b, NS3, E2, and core antigens. The median sum of anti-HCV-secreting B cells to all HCV antigens tested was significantly higher in 39 patients with chronic hepatitis C (47.3 spot forming cells [SFCs]/10(6) peripheral blood mononuclear cells [PBMCs]) than in 9 recovered subjects (15.3 SFCs/10(6) PBMCs; P = .05) or 11 uninfected controls (5.3 SFCs/10(6) PBMCs; P < .001); the significant difference (P = .018) in chronic versus recovered patients was in reactivity to nonstructural antigens NS3 and NS5b. Anti-HCV immunoglubulin M (IgM)-secreting B cells were also readily detected and persisted decades into HCV infection; there was no difference in IgM-positive cells between chronic and recovered patients. ELISpot reactivity to genotype 1-derived antigens was equivalent in patients of genotypes 1, 2, and 3. There was significant correlation between the numbers of anti-HCV IgG-secreting B cells and serum aminotransferase and to the level of circulating antibody. In conclusion, ELISpot assays can be adapted to study B-cell as well as T-cell responses to HCV. Measurement at the single-cell level suggests that humoral immunity plays a minor role in recovery from HCV infection and that B-cell immunity is strongest in those with persistent infection.