Magnitude of CD8+ T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8⁺ T-cell responses and the clinical course of acute HCV infection.
Methods:  Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8⁺ T-cell responses was performed using an interferon-γ-based enzyme-linked immunospot assay using peripheral CD8⁺ T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b.
Results:  Five patients presented detectable HCV-specific CD8⁺ T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8⁺ T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8⁺ T-cells correlated with maximum serum alanine aminotransferase level during the course ( r  = 0.841, P  = 0.036).
Conclusions:  HCV-specific CD8⁺ T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8⁺ T-cell responses, but without development of antibody against HCV.     

Association of multispecific CD4(+) response to hepatitis C and severity of recurrence after liver transplantation.

BACKGROUND & AIMS: After liver transplantation for hepatitis C virus (HCV), reinfection of the allograft invariably occurs. Indirect evidence suggests that the cellular immune response may play a central role. The purpose of this analysis was to determine the correlation between HCV-specific peripheral CD4(+) T-cell responses and the severity of recurrence after liver transplantation. METHODS: Fifty-eight HCV-seropositive patients, including 43 liver transplant recipients with at least 1 year of histological follow-up, were studied. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood and stimulated with either recombinant HCV antigens (core, E2, NS3, NS4, and NS5) or control antigens. RESULTS: Fourteen (40%) of 35 patients with mild or no evidence of histological recurrence within their allografts responded to at least 1 of the HCV antigens. Eleven responded to NS3, 5 to all the nonstructural antigens, and 3 to the HCV core polypeptide alone. In contrast, in the 8 patients with severe HCV recurrence, no proliferation in response to any of the HCV antigens was seen (P = 0. 03) despite responses to the control antigens. CONCLUSIONS: Despite immunosuppression, HCV-specific, major histocompatibility complex class II- restricted CD4(+) T-cell responses are detectable in patients with minimal histological recurrence after liver transplantation. In contrast, PBMCs from patients with severe HCV recurrence, despite being able to proliferate in response to non-HCV antigens, fail to respond to the HCV antigens. These findings suggest that the inability to generate virus-specific T-cell responses plays a contributory role in the pathogenesis of HCV-related graft injury after liver transplantation. It is hoped that further characterization of the immunoregulatory mechanisms related to recurrent HCV will provide the rationale for novel therapeutic strategies and diminish the incidence of inevitable graft loss.     

Strategies for loading dendritic cells with hepatitis C NS5a antigen and inducing protective immunity

Summary.  Dendritic cell (DC)-based vaccination strategies are promising for the treatment of cancers and infectious diseases including hepatitis C virus (HCV). As the induction of T cell-mediated immune responses by DC vaccination is highly dependent on efficient antigen loading of the DCs, the purpose of this study was to identify an optimal nonviral DC loading strategy for HCV NS5a. Furthermore, the efficacy of immunization with the NS5a-loaded DCs in comparison to plasmid encoding NS5a and NS5a protein was evaluated. Transfection of DCs with mRNA was most efficient with close to 100% of DCs expressing NS5a, whereas approximately 10% of protein-pulsed DCs and <1% of plasmid-transfected DCs expressed NS5a, suggesting remarkably different loading efficiencies. Vaccination of mice with NS5a mRNA-transfected DCs or NS5a protein-pulsed DCs resulted in significantly stronger CD4⁺ and CD8⁺ T-cell responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5, in comparison to vaccination with NS5a DNA-transfected DCs, plasmid encoding NS5 or rNS5a protein formulated with alum. Furthermore, vaccination with NS5a mRNA-transfected DCs was superior to vaccination with rNS5a-pulsed DCs. These data have important clinical implications, with mRNA-transfected DCs providing a safe and effective vaccination strategy against hepatitis C and possibly other pathogens.     

Assessment of hepatitis C virus RNA and genotype from 6807 patients with chronic hepatitis C in the United States

Hepatitis C virus (HCV) RNA status and HCV genotype have become important tools in the diagnosis and monitoring of therapy in chronic HCV infection. To establish a database with respect to HCV genotype and serum HCV RNA concentrations in chronic hepatitis C patients in the United States, we analysed 6807 chronic hepatitis C patients who had HCV RNA and HCV genotype tests conducted at a central laboratory. The HCV RNA concentration cut-off for the lower 25th percentile of this population (low titre) was 0.9 × 10⁶ copies ml^–1. The median HCV RNA concentration was 3.5 × 10⁶ copies ml^–1 and the cut-off for the upper 25th percentile (high titre) was 5 × 10⁶ copies ml^–1. Male patients had a median HCV RNA concentration of 3.9 × 10⁶ copies ml^–1, which was significantly higher than the median HCV RNA level for females (2.75 × 10⁶ copies ml^–1; P  < 0.001). HCV genotype 1 was detected in 73% of patients; genotype 2 in 14%; genotype 3 in 8%; mixed genotype in 4%; and genotypes 4, 5 and 6 with a frequency of < 1%. Patients from the Northeast, Southeast and Midwest had significantly ( P  < 0.001) more infections with genotype 1 than patients from the Western and Southern regions. African–American patients were more likely to be infected with genotype 1 when compared with Caucasian, Hispanic or Asian Pacific Islanders ( P  < 0.001). Patients infected with HCV genotype 1 and mixed HCV genotypes had significantly higher serum HCV RNA concentrations when compared with HCV genotypes 2 and 3 ( P  < 0.001 for all comparisons).     

Hepatic inflammatory cytokine mRNA expression in hepatitis C virus-human immunodeficiency virus co-infection

Summary.  Although epidemiologic studies have documented that hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients have accelerated fibrogenesis, especially those with CD4⁺ cell counts <200 cells/mm³, the pathogenic mechanisms are poorly understood. We investigated whether severe immunodeficiency in co-infection is associated with changes in intrahepatic inflammatory cytokine mRNA levels. We measured interferon (IFN)-γ, tumour necrosis factor-α, transforming growth factor (TGF)-β₁, interleukin (IL)-4, IL-10, IL-12p35 and IL-12p40 mRNA levels by real-time PCR performed on liver samples from HCV mono-infected ( n  = 19) and HCV/HIV co-infected ( n  = 24) patients. Co-infected patients had decreased intrahepatic mRNA levels of IFN-γ ( P  = 0.09), IL-4 ( P  = 0.05) and IL-12p35 ( P  = 0.04) compared with mono-infected patients, while IL-10 was increased ( P  = 0.07). In co-infected patients, IFN-γ mRNA levels increased linearly with increasing peripheral CD4⁺ cell counts by 1.23 times relative to the calibrator for every 100 CD4⁺ cells/mm³ increase ( P  = 0.02). No other cytokines were significantly associated with CD4⁺ cell counts. In conclusion, HIV-induced lymphopenia may result in hepatic inflammatory cytokine suppression in HCV/HIV co-infection. Intrahepatic IFN-γ levels are significantly reduced in patients with advanced immunodeficiency. Further studies are needed to assess whether decreased IFN-γ secretion by HCV-specific CD4⁺ cells may account for accelerated fibrogenesis in these patients.     

Biological properties of interleukin-12 and its therapeutic use in persistent hepatitis B virus and hepatitis C virus infection

Summary. Interleukin (IL)-12 is a pleiotropic cytokine produced by antigen-presenting cells in response to diverse stimuli. IL-12 is a key molecule in the regulation of host's immune responses. In particular, IL-12 influences the balance between the T-helper cells type 1 (TH₁) and type 2 (TH₂); it modulates macrophage responses through the control of interferon-gamma synthesis by TH₁ cells; and, suppresses IgE class antibody production (has a suppressive effect on allergic reactions) and promotes a shift in the IgG subclasses. IL-12 enhances resistance to several infectious diseases, is a powerful antitumor agent in vivo , and acts as a vaccine adjuvant. The biological properties of IL-12 point to the potential therapeutic use in persistent hepatitis B virus and hepatitis C virus infection.     

Characterization of the immune response against hepatitis C infection in recovered,and chronically infected chimpanzees

summary . The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti-HCV antibody responses in chronically HCV-infected and recovered chimpanzees. Quantitative measurement of anti-HCV antibody in HCV-infected chimpanzees revealed that the response in HCV- recovered chimpanzees peaked within 4–20 weeks. In contrast, the anti-HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100–200 weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the ³H-uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I-restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9–10 mer overlapping peptides covering the core and NS3 proteins, and IFN-γ ELISPOT technique. Our data indicated early and broad class-I restricted core, and NS3 protein epitope recognitions in HCV-recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8 weeks) were no longer recognized later in infection (followed up to 64 weeks). Cytokines profiling revealed a 50-fold increase in TNF-α secretion in the supernatant of core-specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees.     

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.     

High prevalence of autoimmune phenomena in hepatitis C virus antibody positive patients with lymphoproliferative and connective tissue disorders

The aim of this study was to investigate whether hepatitis C virus (HCV) may perturb the immune system towards autoreactivity. We studied the relationship between the prevalence of anti-HCV and the presence of laboratory and/or clinical autoimmune features in 300 patients: lymphoid malignancies (167) and autoimmune disorders (connective tissue diseases 100; idiopathic thrombocytopenic purpura (ITP) 33). As a control, hepatitis B surface antigen (HBV) and anti-hepatitis B core antigen (anti-HBc) were related to the same parameters.   Anti-HCV and anti-HBV were detected in 68/300 (22.6%) and 70/300 (24.6%) patients, respectively. HCV prevalence was 18% in lymphoproliferative disorders (anti-HBc 28.1%) and 26% in connective tissue disease (anti-HBc 16.3%). Among ITP cases, 12/33 (36.4%) were anti-HCV⁺ and 10/33 (30.3%) anti-HBc⁺. In 24/30 (80%) anti-HCV⁺ patients with lymphoproliferative disorders at least one serologic or clinical autoimmune abnormality was detected. To the contrary, only 10/45 (22.2%) anti-HBc⁺ patients with lymphoproliferative disorders had at least one serologic or clinical abnormality ( P  < 0.0001). A statistically significant correlation was observed between HCV prevalence and the number of autoimmune alterations in both lymphoproliferative and connective tissue disorders, which was not found for anti-HBc.   These data suggest that HCV may skew the immune system toward the production of autoantibodies and also support the possibility that some cases of ITP may be linked to both HCV and HBV infection.     

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.     

慢性丙型肝炎患者外周血单个核细胞中HCV RNA和NS5抗原的检测

目的 研究丙型肝炎病毒（ＨＣＶ）感染外周血个核细胞（ＰＢＭＣ）的情况及其意义。方法 运用非核素原位杂交法（ＮＩＳＨ）和抗生蛋白链菌素－生物素法（ＳＡＢＣ）分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ ＲＮＡ和ＮＳ５抗原。结果 ２０例患者中有８例（４０％）,ＰＢＭＣ中ＨＣＶ ＲＮＡ呈阳性,其中６例（３０％）ＰＢＭＣ中ＮＳ５抗原同时呈阳性。ＨＣＶＲＮＡ主要分布于胞浆中,而ＮＳ５抗原还可以出现在胞膜     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒检测及其意义

目的 探讨外周血单个核细胞（ＰＢＭＣｓ）在丙型肝炎病毒（ＨＣＶ）的感染中的作用。方法 对２２例慢性丙型肝炎患者２１例抗－ＨＣＶ（＋）血液管析患者及１２例健康献血员的ＰＢＭＣｓ分别进行ＨＣＶＲＮＡ,ＨＣＶ抗原检测及电镜观察。结果 （１）２２生丙型肝炎肝炎患者ＰＢＭＣｓ中有７７．３％（１７／２２）ＨＣＶＲＮＡ阳性,（２）感染ＨＣＶ的ＰＢＭＣｓ中电镜下发现复制的ＨＣＶ颗粒;（３）ＨＣＶ颗粒阳笥者的血清和     

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.     

Low-level hepatitis C viremia and humoral immune response to NS4 in chronic hepatitis B virus-hepatitis C virus coinfection

BACKGROUND: There is a limited amount of published data on the interference of hepatitis B virus (HBV) on hepatitis C virus (HCV). The aim of this study was to investigate the effect of concurrent HBV infection on serum titers of HCV RNA and HCV antibody profiles in chronic HCV infection. METHODS: The clinical and virological profiles (serum titers of HCV RNA, HCV genotypes and antibody profiles) of 25 patients with chronic HBV-HCV coinfection were compared with those of 25 age- and sex-matched patients with HCV infection alone. RESULTS: Among the 25 patients with HBV-HCV coinfection, only 3 were found hepatitis Be antigen (HBeAg) and HBV DNA positive by hybridization assays, and the other 11 were found HBV DNA positive by polymerase chain reaction. Genotype 1b was dominant in both HBV-HCV coinfection and HCV infection alone (64% versus 84%, P > 0.1). Patients with HBV-HCV coinfection had significantly lower alanine aminotransferase (ALAT) levels and inflammatory scores but higher fibrosis scores than those with HCV infection alone. Serum titers of HCV RNA were significantly lower in HBV-HCV coinfection than in HCV infection alone. The frequency and relative intensity of antibody response to core, E2/NS1, NS3, and NS5 showed no significant difference between the two groups, but antibody response to NS4 was diminished significantly in HBV-HCV coinfection. CONCLUSIONS: In HBV-HCV coinfection, serum levels of HBV DNA are usually low or undetectable. Concurrent HBV infection, however, could interfere with HCV replication and suppress antibody response to NS4. The biological significance of selective inhibition of humoral immune response to NS4 in HBV-HCV coinfection should be further studied.     

慢性丙型肝炎患者外周血单个核细胞丙型肝炎病毒感染研究

目的 以分子生物学、免疫学有电镜等技术对慢性丙型肝炎患者ＰＢＭＣｓ进行综合检测,以证实其受ＨＣＶ感染,并试图在电镜下发现和证实感染细胞内ＨＣＶ颗粒。方法 对逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学等技术分别检测患者ＰＢＭＣｓ内的ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,以常规和免疫电镜技术观察研究感染细胞内ＨＣＶ的形态、定位和超微结构。结果 ＨＣＶ ＲＮＡ阳性检出率为７７．２％（１７／２２）,ＨＣＶＡｇ     

Lymphoproliferative responses to hepatitis C virus core,E1, E2, and NS3 in patients with chronic hepatitis C infection treated with interferon alfa

The quality of the hepatitis C virus (HCV)-specific T-cell response may greatly determine the course of an HCV infection. An adequate T-cell response may contribute to a successful clearance of the virus and a rapid recovery from the disease. An inadequate response may lead to viral persistence and may eventually contribute to the pathogenesis of hepatocellular damage in chronic disease. The effect of interferon alfa (IFN-alpha), presently the most popular therapeutic agent for chronic HCV infections, on HCV-specific T-cell responses is completely unknown. To demonstrate the presence of HCV-specific T lymphocytes during chronic HCV infections, to know their antigenic specificities, and to examine possible effects of IFN-alpha treatment on their presence and antigen recognition patterns, we have stimulated peripheral blood mononuclear cells (PBMC) from 35 chronic HCV patients with nine pools of synthetic peptides representing the HCV Core, E1, and E2 proteins as well as with a recombinant NS3 protein. The proliferative responses of PBMC from 16 healthy control subjects toward these antigens were measured for comparison. Lymphoproliferative responses of patients with chronic HCV infections were assayed either before (in 10 patients), during (in 13 patients), or after (in 21 patients) treatment with IFN-alpha. The analysis showed that PBMC from most HCV patients consistently recognized the COOH-terminal part of the core protein. E1, E2, and NS3 were recognized less frequently. This recognition pattern was not related to the therapy with IFN-alpha nor to the clinical response of the patient toward this therapy. The response to the Core protein could be fine-mapped to the COOH-terminal region encompassing amino acids (aa) 73 to 92, 121 to 140, 145 to 164, and 157 to 176. (Hepatology 1996 Jan;23(1):8-16)     

Studies of interleukin-12 in chronic hepatitis B virus infection

Summary. Interleukin-12 is produced by antigen-presenting cells and regulates the balance between TH₁/TH₂ lymphocyte subsets, promoting cell-mediated immune reactions. Amongst patients with chronic hepatitis B undergoing interferon-alpha treatment, only those who clear hepatitis B virus show a substantial increase in the production of biologically active IL-12 and an inverse ratio between serum levels of IL-12p40 subunit and IL-12. The peak of serum IL-12 occurs after the hepatitis flare and precedes or coincides with the time of HBe seroconversion. These data indicate that IL-12 is an important element for establishing the host immune control on hepatitis B virus replication in patients with chronic hepatitis B virus infection.     

Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species

Impaired T-cell responses in chronic hepatitis C virus (HCV) patients have been reported to be associated with the establishment of HCV persistent infection. However, the mechanism for HCV-mediated T-cell dysfunction is yet to be defined. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in suppressing T-cell responses. In this study we examined the accumulation of MDSCs in human peripheral blood mononuclear cells (PBMCs) following HCV infection. We found that CD33(+) mononuclear cells cocultured with HCV-infected hepatocytes, or with HCV core protein, suppress autologous T-cell responses. HCV core-treated CD33(+) cells exhibit a CD14(+) CD11b(+/low) HLADR(-/low) phenotype with up-regulated expression of p47(phox) , a component of the NOX2 complex critical for reactive oxygen species (ROS) production. In contrast, immunosuppressive factors, arginase-1 and inducible nitric oxide synthase (iNOS), were not up-regulated. Importantly, treatment with an inactivator of ROS reversed the T-cell suppressive function of HCV-induced MDSCs. Lastly, PBMCs of chronic HCV patients mirror CD33(+) cells following treatment with HCV core where CD33(+) cells are CD14(+) CD11b(+) HLADR(-/low) , and up-regulate the expression of p47(phox). CONCLUSION: These results suggest that HCV promotes the accumulation of CD33(+) MDSC, resulting in ROS-mediated suppression of T-cell responsiveness. Thus, the accumulation of MDSCs during HCV infection may facilitate and maintain HCV persistent infection.