Concomitant augmentation of type 1 CD4+ and CD8+ T-cell responses during successful interferon-alpha and ribavirin treatment for chronic hepatitis C virus infection

The combined interferon-alpha (IFN-alpha) and ribavirin (IFN-alpha/ribavirin) therapy for chronic hepatitis C virus (HCV) infection results in sustained viral eradication in 31%-64% of the patients. Previous studies have strongly suggested that HCV-specific T-cell responses maybe modulated during this therapy. The objective of this study was to further define the effect of IFN-alpha/ribavirin therapy on type 1 and type 2 HCV-specific CD4(+) and CD8(+) T-cell responses during IFN-alpha/ribavirin therapy. Toward this, serial CD8(+) T-cell responses to HCV-derived epitopes and CD4(+) T-cell responses to the HCV core antigen were analyzed in four patients before (baseline), during (at 24 weeks), and at the end (at 48 weeks) of IFN-alpha/ribavirin therapy. Therapy-induced viral clearance in three patients was associated with a significant augmentation of HCV-specific type 1 CD4(+) and CD8(+) T-cell responses. In contrast, in a patient who did not respond to therapy, a significant HCV-specific CD4(+) Th2 cell reactivity was observed accompanied by a lack of augmentation of the HCV-specific CD8(+) T-cell reactivity. These results indicate that enhancement of HCV-specific CD4(+) and CD8(+) T-cell responses is an important factor in determining the response to the IFN-alpha/ribavirin therapy and the outcome of the HCV infection.     

Priming and stimulation of hepatitis C virus-specific CD4+ and CD8+ T cells against HCV antigens NS4, NS5a or NS5b from HCV-naive individuals: implications for prophylactic vaccine

Hepatitis C virus (HCV) is a devastating human pathogen, yet there is no vaccine available for this virus. From studies with acute or chronic HCV-infected humans and chimpanzees, T-cell responses against HCV-derived conserved non-structural antigens have been correlated with viral clearance. In this study, recombinant adenoviral vectors containing HCV-derived NS4, NS5a or NS5b genes were employed to endogenously express the HCV antigens in human dendritic cells (DCs). The DCs expressing these HCV antigens exhibited normal phenotype and function. Intriguingly, we found that the DCs expressing HCV NS4, NS5a or NS5b antigens were able to significantly stimulate autologous T cells obtained from uninfected healthy individuals. These T cells produced various cytokines and proliferated in an HCV antigen-dependent manner. Evidence of both CD4(+) and CD8(+) T-cell responses generated in vitro against HCV NS4, NS5a or NS5b were obtained. HCV NS4 was much less stimulatory for CD4(+) and CD8(+) T cells than NS5. Further, in secondary assays, the CD4(+) T cells primed in vitro exhibited HCV antigen-specific proliferative responses against recombinant protein antigens. In summary, we provide conclusive evidence of in vitro stimulation of CD4(+) and CD8(+) T cells from HCV-naive individuals against HCV antigens NS4, NS5a and NS5b. The studies with naive T cells represent early events in the induction of cellular immune responses, which most likely govern the outcome of HCV infection. These studies have significant implications in designing vaccines for HCV infection in both prophylactic and therapeutic settings.     

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.     

Lipidation of T helper sequences from hepatitis C virus core significantly enhances T-cell activity in vitro

Successful elimination of the hepatitis C virus (HCV) during acute infection has been linked to strong HCV-specific in vitro T-cell proliferation, whereas T cells from patients with chronic hepatitis C respond only weakly to HCV antigens. Lipid-coupled peptides are immunostimulants, which might provide a basis for novel therapeutic strategies against HCV. Therefore, in 20 patients with chronic hepatitis C, we studied whether tri-palmitoyl-S-cysteine-coupled peptides could modify in vitro T-cell proliferation (by [3H]thymidine uptake) in response to virus core and NS4. The lipopeptides corresponded to five immunodominant T helper epitopes of HCV core. Contrary to unmodified peptides, the lipopeptides specifically enhanced [3H]thymidine uptake in response to HCV antigens but not to a non-HCV related control antigen. They increased the frequency of responders (stimulation index, SI > or = 4) to core (13/20 versus 2/20; p = 0.0008) and NS4 (20/20 versus 7/20; p < 0.0001) among our patients with chronic hepatitis C. This immunostimulatory effect was dose-dependent, and was observed specifically with lipopeptides corresponding to the HCV epitopes. Our data demonstrate that the poor in vitro T-cell proliferation of patients with chronic hepatitis C can be improved when T cells are co-stimulated with HCV core-derived T helper lipopeptides, while the same peptides in unlipidated form had no effects. Thus, lipopeptides corresponding to HCV T-cell epitopes may offer novel immunomodulatory strategies against HCV.     

Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+ response in patients with chronic hepatitis C

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.     

Interferon-gamma is produced by CD8 T cells in response to HLA-A24-restricted hepatitis C virus epitopes after sustained virus loss

Differences in cytotoxic T lymphocyte activity in hepatitis C virus infection may account for the outcome of interferon monotherapy. To investigate this hypothesis, we analysed the response of peripheral CD8(+) T cells that recognized epitopes presented by HLA-A*2402. We synthesized HLA/beta2-microglobulin/peptide complexes using two epitopes. Production of interferon-gamma by CD8(+) T cells in response to plastic-bound monomeric HLA/peptide complex was observed frequently in sustained virus responders (SVR) (n = 13) against all the peptides, NS31296-1304 (the percentage of responding patients, 61.5%) and core 129-137 (53.8%), while no interferon-gamma production was observed in non-responders (NR) (n = 13) for any of the peptides. Tetramer-staining showed the presence of CD8(+) T cells specific for all the peptides except NS31296-1304 in two SVR at the end of interferon monotherapy, although hardly any such cells were found in four NR. Specific killing was observed against peptides NS31296-1304 (3/4) and core 129-137 (1/4) in sustained responders but none in non-responders. These results suggest that the responses of cytotoxic T lymphocytes (CTLs) were induced during interferon therapy in these patients and that interferon-gamma production by CD8(+) T lymphocytes against HCV NS31296-1304 and core 129-137 are well maintained in patients with SVR compared with those with NR. These findings emphasize the importance of the CD8(+) T cell response in controlling HCV infection.     

The use of class-I HLA tetramers for the detection of hepatitis C virus NS3-specific CD8(+) T cells in patients with chronic infection

BACKGROUND/AIMS: New methods to detect virus-specific T-cell responses have recently been developed. Several human leukocyte antigen (HLA)-peptide tetramers for the detection of hepatitis C virus (HCV)-specific CD8(+) T cells are under evaluation. METHODS: Evaluation of one HLA class I-tetramer (HCVNS3-2) for the detection of HCV NS3-specific CD8(+) T cells in a series of 38 HLA-A2(+) chronically infected patients. RESULTS: Almost half (42%) of the patients had detectable NS3-specific CD8(+) T cells. The frequencies of such cells ranged from 0.01% to 0.22% of total CD8(+) T cells. No significant differences in clinical features or mean viral load were detected between patients with or without tetramer + CD8(+) T cells. CONCLUSIONS: The tetramer HCVNS3-2 may be very useful for the study of the HCV-specific CD8(+) immune response. Combination of this reagent with other tetramers based on other HCV peptides may help in the understanding of the immune response to the virus. However, a panel of tetramers based on several parts of the HCV polyprotein may be a mandatory requirement to explore the whole breadth of the CD8(+) T-cell response against HCV and to detect that response in the majority of patients with chronic infection.     

Functionally distinct helper T-cell epitopes of HCV and their role in modulation of NS3-specific,CD8+/tetramer positive CTL

Hepatitis C virus specific (HCV-specific) CD8+ cytotoxic T cells play a critical role in viral clearance. Low HCV-specific cytotoxic T lymphocyte (CTL) responses in chronic HCV infection may favor the persistence of virus, whereas stimulation and expansion of HCV-specific CTL activity may assist elimination of HCV infection. Helper T cells control the intensity of CD8+ T-cell responses and helper T-cell responses are known to be compromised in chronic carriers of HCV. In this study, we wanted to ascertain if strengthening the Th response could increase the intensity of CTL activity against HCV target antigens. We selected a synthetic CTL peptide NS3(1073-1081)), two Th1 epitopes, peptide NS3(358-375) and NS5B(155-172), and one Th2 epitope, peptide NS3(505-521). By using the four peptides alone or in combinations, we stimulated peripheral blood cells isolated from a chronic hepatitis C patient in vitro and then analyzed CD8 T cells specific for the NS3(1073-1081) CTL epitope in A2 tetramer staining and cytotoxicity assays. The results demonstrated that CTL responses could be augmented by helper T-cell epitopes NS3(358-375) and NS5B(155-172). Th2 epitope NS3(505-521) inhibited augmentation of CTL activity by Th1 epitopes. This inhibitory effect could be overcome by combining the two Th1 epitopes NS3(358-375) and NS5B(155-172) together with NS3(505-521). Under such conditions, CTL frequency was restored, but cytotoxic activity remained low suggesting that the help provided under these cultures was sufficient to drive proliferation of CTL, but not sufficient to drive differentiation into mature killer cells. These results may provide some insights into compromised CTL activity in HCV viral persistence.     

Influence of HCV genotype and co-infection with human immunodeficiency virus on CD4(+) and CD8(+) T-cell responses to hepatitis C virus

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.     

Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection

Hepatitis C virus (HCV) RNA persistence in the liver has been described even after apparent resolution of HCV infection. Because T-cell reactivity plays a role in recovery from HCV infection, virus-specific T-cell responses were investigated in apparently recovered individuals in whom hepatic HCV RNA persistence was documented: 15 sustained virological responders to interferon (IFN)-treatment and 9 asymptomatic aviremic anti-HCV carriers. HCV-specific CD4(+) T-cell proliferative responses were detected significantly more often in apparently recovered individuals (sustained virological responders: 60%; asymptomatic anti-HCV carriers: 66%) compared with 50 chronic hepatitis C patients (28%; P < 0.05). However, T-cell frequencies and numbers tended to decline over time and the number of HCV proteins targeted by CD4(+) T-cell proliferative responses was limited. Interestingly, liver viral load correlated inversely with virus-specific immune responses. Thus, CD4(+) T-cell responders showed significantly lower hepatic HCV RNA levels (P < 0.05). HCV-specific IFN-gamma-secreting CD4(+) T-cells were not detected in all the apparently recovered patients although they were found significantly more often compared with chronic hepatitis C patients (P < 0.05). Also, HCV NS3-specific CD8(+) T-cells were detected in 11 HLA-A2-positive apparently recovered individuals (8 sustained virological responders and 3 asymptomatic anti-HCV carriers); T-cell frequencies tended to be greater in those patients who had lower hepatic viral levels. In conclusion, HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist in the liver indicating that HCV replication may be prolonged in the face of an insufficient or inadequate virus-specific CD4(+) and CD8(+) T-cell response.     

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.     

HLA class II alleles and chronic hepatitis C virus infection

The aim of this study was to investigate association of human leucocyte antigens (HLA)-DRB1 and DQB1 polymorphisms with hepatitis C virus (HCV) infection and with the occurrence of severe liver fibrosis/cirrhosis in chronically infected patients. Ninety-nine white patients, from southeast Brazil, with confirmed HCV chronic infection were included in the study. Severe fibrosis/cirrhosis (METAVIR scores F3-F4) was present in 49 patients. HLA-DRB1 specificities and DRB1*11 and DQB1* alleles were determined by PCR-SSP, and their frequencies were compared between patients and a control group of 103 healthy white Brazilian individuals. The results confirmed previous reports of the association of DRB1*11 and DQB1*03 with protection from chronic HCV infection, but did not confirm their association with protection from severe fibrosis/cirrhosis. Furthermore, the results suggested that the polymorphic sites on HLA molecules responsible for protection from chronic HCV infection are encoded not only by the DRB1*1101 and DQB1*0301, as suggested in the literature, but also by other DRB1*11 and DQB1*03 alleles. Thus, we hypothesized that the common polymorphic residues shared by different DRB1*11 and/or DQB1*03 alleles might be responsible for selection of viral epitopes for presentation to CD4(+) T cells, leading to an efficient immune response against the virus.     

Induction of potent cellular immune response in mice by hepatitis C virus NS3 protein with double-stranded RNA

Double-stranded RNA is produced during virus replication and, together with the viral antigen, is responsible for inducing host antivirus immunity. The hepatitis C virus (HCV) non-structural protein-3 (NS3) has been implicated in the immune evasion of HCV, and is one of the prime targets for inducing immunity against HCV infection. Mice were immunized with recombinant NS3 protein (rNS3) and poly (I:C) emulsified in Montanide ISA 720 (M720). Cytokine production was assayed by enzyme-linked immunospot assay, and CD4(+) IFN-gamma(+) T helper (Th) cells or CD8(+) IFN-gamma(+) cytotoxic T lymphocytes were detected by flow cytometry. Anti-NS3 titre and immunoglobulin G2a (IgG2a) and IgG1 levels were monitored by enzyme-linked immunosorbent assay. Administration of rNS3 formulated in poly (I:C) and M720 induced anti-NS3 titres with a predominantly IgG2a isotype comparable to those induced by rNS3 in CpG-ODN and M720. The cytokine profiles showed that this formulation induced a Th1-biased immune response with several-fold more interferon-gamma (IFN-gamma)-producing cells than interleukin-4-producing cells. In contrast, rNS3 in M720 induced a Th2-biased immune response. The frequency of IFN-gamma-producing CD4(+) and CD8(+) cells induced by rNS3 in poly (I:C) and M720 was significantly higher than that induced by rNS3, rNS3 in M720, or rNS3 in poly (I:C), and was comparable to that induced by rNS3 in CpG-ODN with M720. The antigen-specific CD8(+) T-cell immune response persisted for up to 7 months after immunization. In conclusion, poly (I:C) with rNS3 in M720 can elicit a strong and persistent Th1-biased immune response and a cytotoxic T-lymphocyte response through cross-priming in mice. This study highlighted a promising formulation for inducing an efficient cellular immune response against HCV that has potential for HCV vaccine development.     

Differential expansion of T-cell receptor variable beta subsets after antigenic stimulation in patients with different outcomes of hepatitis C infection

Persistent antigenic stimulation during chronic hepatitis C may alter the T-cell receptor variable chain beta (TCR BV) repertoire as well as the cytokine responses of hepatitis C virus (HCV)-specific T lymphocytes. We analysed the distribution of the TCR BV subsets 2.1, 3.1, 5.1, 6.1, 8, 13.1, 13.6, 14.1, 17.1, 21.3 in relation to intracytoplasmic expression of interleukin-2, interferon-gamma, interleukin-4 and interleukin-10. Using flow cytometry, CD45RO+ memory T cells of 27 patients with chronic hepatitis C, eight patients with resolved HCV infection and 16 non-HCV-related controls were studied with and without stimulation by the HCV core, NS3, NS4, NS5a and NS5b proteins. Patients with chronic and resolved hepatitis C differed by larger basal TCR BV2.1+, BV6.1+, BV17.1+ and BV21.3+ subsets in chronic hepatitis C, which were correlated to the numbers of T cells with spontaneous interleukin-2 and interferon-gamma production (r=0.51-0.73, P<0.05). Upon HCV-specific stimulation these subsets did not expand, whereas a marked in vitro expansion of TCR BV8+ T cells in response to all HCV proteins was selectively noted in chronic hepatitis C (P<0.05). This expansion of TCR BV8+ memory T cells was significantly correlated to HCV-induced interleukin-10 expression (r=0.58-0.98, P<0.01). Thus, differential involvement of selected TCR BV subsets may be related to the outcome of HCV infection.     

Prospective study of viral clearance and CD4(+) T-cell response in acute hepatitis C primary infection and reinfection.

BACKGROUND: The outcome of acute hepatitis C is determined by early host-virus interactions, particularly involving the antiviral T-cell response. OBJECTIVES: To identify early prognostic markers of spontaneous resolution of acute hepatitis C by performing a comprehensive analysis of viral and immunological factors during the natural course of acute HCV infection and reinfection. STUDY DESIGN: 20 patients were investigated prospectively during acute HC or confirmed reinfection and 18 of them during follow up after spontaneous or treatment-induced elimination of the virus and resolution of the disease. Multiparameter flow cytometry was used to functionally characterize virus-specific CD4(+) T-cell responses relative to the virologic outcome. RESULTS: Parallel immunologic and virologic monitoring of patients with acute HC identified distinct patterns of host-virus interaction related to HCV persistence or clearance. The highest frequency of antiviral Th1 cytokine-producing CD4(+) T-cells was observed in patients with HCV reinfection, preceding rapid viral clearance within 3 weeks after disease onset. In all patients who subsequently cleared viremia, CD4(+) T-cells produced Th1 cytokines following stimulation with non-structural HCV antigens (NS3 and NS4). In contrast, a chronic course of disease was associated with the absence of antiviral Th1 cytokine producing cells from the first weeks after onset of disease (acute persistent HC), or with fluctuating RNA levels (yo-yo pattern) and gradual waning of antiviral Th1 cells. CONCLUSIONS: The results highlight the variability of immune response pattern in acute hepatitis C. Most importantly, "acute persistent hepatitis C" and a lack of TH1 effector cells within the first months of acute hepatitis C represent efficacious predictors of viral persistence and could thus be used as criteria in selecting candidates for early antiviral treatment.     

Oligoclonal CD8+ T-Cell Expansion in Patients with Chronic Hepatitis C Is Associated with Liver Pathology and Poor Response to Interferon-α Therapy

The role of CD8(+) T lymphocytes in chronic hepatitis C virus (HCV) infection and in liver injury with subsequent development of fibrosis and cirrhosis is poorly understood. To address this question, we performed a follow-up study including 27 chronically HCV-infected individuals. We determined clonality and phenotypes of circulating CD8(+) T cells employing TCRBV spectratyping. Antigen specificity was tested by rMHC-peptide tetramer staining and stimulation with recombinant HCV antigens. In addition, T-cell clonality and phenotypes were followed during the variable clinical response of interferon- (IFN) alpha treatment. We could demonstrate that CD8(+) T-cell expansions were significantly associated with liver fibrosis and cirrhosis. Likewise, increased oligoclonality of circulating CD8(+) T cells in chronic HCV infection was identified as an indicator for poor clinical response to IFN-alpha therapy. Moreover, we also found that IFN-alpha therapy enhanced the differentiation of CD8(+) T cells towards a late differentiation phenotype (CD28(-) CD57(+)). In cases of virus elimination the disappearance of expanded terminally differentiated CD8(+) cells was observed. Thus, this study identifies an association of clonal expansions of circulating CD8(+) T cells with liver pathology and provides a possible explanation for the fact that response to IFN-alpha therapy diminishes with the duration of infection.     

丙型肝炎病毒非结构区5个细胞毒性T细胞表位的鉴定

目的 采用T细胞表位预测软件结合体外实验鉴定丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位.方法 采用T表位预测软件Rankpep预测HCV特异性CTL表位,选择候选CTL表位加以合成;用候选CTL表位肽分别刺激HCV感染者以及健康志愿者的外周血单个核细胞(PBMC),采用酶联免疫斑点试验(ELISPOT)检测PBMC中肽特异性分泌IFN-γ的斑点形成细胞(spots forming cells,SFC)的水平,采用细胞内细胞因子染色(intracellular cytokine staining,ICS)检测PBMC中肽特异性IFN-γ+CD8+T细胞的水平.结果 用5条候选CTL表位肽[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)和NS5a 367(TVSSALAEL)]分别刺激10个HCV感染者和2个健康者的PBMC后,健康者的PBMC不产生IFN-γ而7个HCV感染者的PBMC产生IFN-γ;HCV感染者的PBMC中肽特异性分泌IFN-γ的细胞的频率为(5-36)SFC/105 PBMC,肽特异性IFN-γ+CD8+T细胞占总CD8+T细胞的百分比为0.02%～0.25%.结论 ELISPOT结果和ICS结果证实5条肽NS3 450、NS3 594、NS4b 78、NSSa 416和NS5a 367为全新的HCV特异性CTL表位.     

Selection of hepatitis B virus variants with aminoacid substitutions inside the core antigen during interferon-alpha therapy

The hepatitis B virus (HBV) core antigen carries many epitopes relevant for B and T cell response that show aminoacid variation during viral infection. In a longitudinal analysis, sequential serum samples of 15 patients that suffered from chronic HBV infection were collected before, during, and after high-dose IFN-alpha treatment. The HBV preCore/Core (preC/C) sequence of the selected samples in each patient was determined and analysed for sequence variations compared to the pretreatment sample. The positions of HBV core aminoacid substitutions were assigned to immunodominant B, CD4(+) and CD8(+) cell epitopes. Seventy-five percent of all aminoacid substitutions were found within immunodominant T and B cell epitopes (12.5% were inside known HBV core mutation cluster regions) that show an increased number of clustered aminoacid substitutions during chronic HBV infection and overlap partially with the immunodominant epitopes. Only 12.5% of the detected core antigen aminoacid substitutions could not be assigned to any epitope or mutation cluster region. Stable HBV core antigen aminoacid substitutions, which were found between pretreatment sequence and the last sequence analysed during therapy, were found most frequently inside T helper cell epitopes. This longitudinal analysis of aminoacid substitutions inside the HBV core antigen in patients with chronic HBV infection shows that core aminoacid variations occur most frequently inside immunodominant HBV core epitopes, possibly due to an immuneselective pressure of the host against the virus. The data also suggest that stable HBV variants with aminoacid substitutions in immunodominant core epitopes can be selected during high-dose IFN-alpha therapy and persist after the end of treatment.     

Histological improvement of chronic liver disease after spontaneous serum hepatitis C virus clearance

The long-term histological and virological outcomes of spontaneous circulating hepatitis C virus (HCV) clearance were studied in chronic liver disease. Between 1979 and 1984, three patients underwent laparoscopy for chronic non-A, non-B liver disease, and two were found to have cirrhosis and one with chronic active hepatitis. After HCV assays became available in 1990, they were positive persistently for HCV antibody without serum HCV RNA. Reductions of antibody levels to HCV core and/or nonstructural proteins were observed, and liver biopsies were undertaken between 1995 and 2000. Liver biopsies at 11-19 years after laparoscopy disclosed marked alleviation of liver inflammation and fibrosis in each case although a low grade of inflammation remained. The two patients with cirrhosis no longer showed histological features of cirrhosis, and the poor liver function in one patient had been ameliorated. Liver specimens from two patients were subjected to polymerase chain reaction to detect positive and negative HCV RNA strands and hepatitis B virus DNA. Only the positive HCV RNA strand was detected for one patient who had previously cirrhosis. Liver specimens were examined from another six nonviremic HCV-seropositive individuals without chronic liver disease. Five patients displayed low-grade liver inflammation without evident fibrosis, but none had any viral genome in the liver. These findings suggest that spontaneous circulating HCV clearance in chronic liver disease confers favorable liver histological outcome, although occult HCV infection persists. J. Med. Virol. 69:41-49, 2003.     

Peptide-loaded chimeric influenza virosomes for efficient in vivo induction of cytotoxic T cells

Virus-specific CD8(+) T cells are thought to play an important role in resolving acute hepatitis C virus (HCV) infection as viral clearance has been associated with a strong and sustained CD8(+) T cell response. During the chronic state of HCV infection virus-specific T cells have a low frequency and a reduced responsiveness. Based on this, a therapeutic vaccine increasing the frequency of specific T cells is a promising alternative for the treatment of chronic HCV infection. We improved an existing vaccine platform based on immunopotentiating reconstituted influenza virosomes (IRIVs) for efficient delivery of peptide epitopes to the MHC class I antigen presentation pathway. IRIVs are proteoliposomes composed of phospholipids and influenza surface glycoproteins. Due to their fusogenic activity, IRIVs are able to deliver encapsulated macromolecules, e.g. peptides to immunocompetent cells. We developed a novel method based on chimeric virosomes [chimeric immunopotentiating reconstituted influenza virosomes (CIRIVs)] combining the high peptide-encapsulation capacity of liposomes and the fusion activity of virosomes. This new approach resulted in a 30-fold increase of the amount of incorporated soluble peptide compared with current preparation methods. To study the immunogenicity of chimeric virosomes HLA-A2.1 transgenic mice were immunized with CIRIVs containing the HCV Core132 peptide. Core132-CIRIVs efficiently induced specific cytotoxic and IFNgamma-producing T cells already with low peptide doses. Vaccine formulations, which include combinations of different HCV-derived CTL epitopes could be used to induce not only a strong but also a multi-specific CTL response, making them potential candidates for therapeutic and maybe prophylactic T cell vaccines in humans.