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.     

Genetic immunization and comprehensive screening approaches in HLA-A2 transgenic mice lead to the identification of three novel epitopes in hepatitis C virus NS3 antigen

Interferon-gamma (IFN gamma)-producing CD8+ T cells have been shown to play a key role in the control or eradication of hepatitis C virus (HCV) infections. In particular, T cells specific of the non-structural protein 3 (NS3) are often associated with control of viremia. The aim of the study was to identify novel HLA-A2 restricted CD8+ T cell epitopes specific of NS3 using a combination of comprehensive approaches. HLA-A2.1 transgenic mice were immunized with a DNA vaccine optimized for NS3 specific epitope presentation and induced CD8+ T cell reactivity was screened using 42 algorithm-predicted peptides as well as a library of 78 overlapping 15-mer peptides spanning the whole protein. Three epitopes mapping within the NS3 protease (GLL: aa 1038-1047) or helicase (ATL: aa 1260-1268 and TLH: aa 1617-1625) were identified. These epitopes, which display similar and high in vitro binding capacities to soluble HLA-A2 molecules, are able to induce either cytotoxic T lymphocytes (CTL) and/or IFN gamma-producing T cells. Comparative in vitro target cell sensitization studies revealed a higher immunogenicity of the GLL peptide as compared with both ATL and TLH peptides. This peptide was capable to recall in vitro HCV-specific IFN gamma and IL-10-producing T cells from peripheral blood mononuclear cells (PBMC) of chronically infected patients. These data increase the pool of NS3-specific CD8+ T cell epitopes available to analyze HCV associated immunity and could contribute to the design and evaluation of candidate vaccines.     

Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b

Cellular immunity against multiple Hepatitis C virus (HCV) proteins is observed in patients acutely infected with HCV most of whom later resolve infection. We wished to assess humoral immunity in patients infected with HCV 1a or 1b genotypes in relation to viral load using plasma samples from HCV-infected individuals and a panel of peptides representing immunodominant epitopes of HCV structural and nonstructural proteins. Plasma from HCV 1a- and 1b-infected patients, respectively, were divided into two groups: patients with low viral load (<==100,000 RNA copies/ml) and patients with high viral load (>/=10,000,000 RNA copies/ml). The antigens were peptides representing epitopes from immunodominant regions of HCV core, E2, NS3, and NS4 proteins, as well as the hypervariable (HVR) epitopes in E2 from genotypes 1a and 1b. Individuals infected with HCV 1a evoked a stronger immune response to many immunodominant epitopes of HCV relative to individuals infected with HCV 1b. Moreover, among individuals infected with HCV 1a, those with low viral loads mounted significantly greater responses against these epitopes than did individuals with high viral loads. Our observations demonstrate that quantitatively different antibody responses are elicited against HCV depending on the genotype of infecting virus, and suggest that humoral immunity directed against multiple immunodominant epitopes in HCV 1a-infected individuals may help lower viral load in vivo.     

Viral escape and T cell exhaustion in hepatitis C virus infection analysed using Class I peptide tetramers

Hepatitis C virus (HCV) has infected over 170 million people world wide, and in the majority sets up a chronic infection associated with hepatic inflammation. How it evades host immunity, particularly CD8+ T cells (CTL) is unclear, but two major factors are likely to operate, viral escape mutation and T cell exhaustion. We have investigated the role of CTL in control of infection during acute disease using Class I peptide tetramers. Although the immune response is quite diverse and numerous epitopes can be targeted, we observe that, especially during acute disease, one epitope (NS3 1073-81) is commonly recognised in HLA-A2 positive individuals. However, the levels of response to this epitope (and others) are very much lower if persistence is established. We examined in detail whether the cause of this low level of reactivity is due to mutation within the epitope. We find that, in fact this epitope is highly conserved during chronic infection, at a clonal level, between individuals, and over time. Thus, although variation within the epitope does occur, lack of reactivity in peripheral blood against this epitope in chronic disease, and loss of control of virus cannot be explained entirely by viral escape. Escape through mutation probably does play an important role in persistence of HCV, but we also discuss other mechanisms which lead to attenuation of T cell responses which may be important in determining the outcome.     

Comparison of cytotoxic T-lymphocyte responses to hepatitis C virus core protein in uninfected and infected individuals.

Cytotoxic T lymphocytes have been implicated in the control of hepatitis C virus (HCV) infection. Recognition by cytotoxic T lymphocytes of epitopes within HCV core protein has been defined previously by in vitro stimulation with synthetic peptides. The aim of this study has been to examine cytotoxic T-lymphocyte responses generated against peptides produced naturally following intracellular processing of viral protein. Antigen-specific cytotoxic T-lymphocyte lines were generated from both HCV uninfected and infected individuals by culturing CD8+ T cells with autologous dendritic cells loaded intracytoplasmically with recombinant HCV core protein. Analysis of the epitopes recognized by core protein-specific cytotoxic T lymphocytes used synthetic peptides that were selected based on their predicted binding to HLA-A*0201 molecules. Core protein-specific cytotoxic T lymphocytes derived from HCV uninfected and infected individuals were able to lyse autologous target cells pulsed with each of 5 predicted epitopes. Generation of HCV-specific cytotoxic T lymphocytes using dendritic cells as antigen presenting cells provides a method of comparing the potential repertoire of cytotoxic T-lymphocyte responses to the responses that occur in chronically infected individuals. No evidence of a qualitatively different response by patient cytotoxic T lymphocytes was apparent which might explain persistence of the virus.     

Lack of optimal T-cell reactivity against the hepatitis C virus is associated with the development of fibrosis/cirrhosis during chronic hepatitis

Chronic hepatitis C virus (HCV) infection develops in 85% of exposed individuals and 20% develop cirrhosis. However, the pathogenesis of this process is not well-understood. The objective of this study was to determine whether HCV-reactive T cells play a role in the process of development of cirrhosis during chronic HCV infection. We analyzed 21 human leukocyte antigen (HLA)-A2 patients with chronic HCV infection (9 with histology of inflammation and 12 with histology of fibrosis/cirrhosis). The frequency of CD8(+) T cells reactive to 12 HCV-derived epitopes was determined by an interferon-gamma enzyme-linked immunospot (ELISPOT) assay. The frequency of CD4(+) Th1 and Th2 cells reactive to the HCV core antigen was determined by interferon-gamma and interleukin-5 ELISPOT assays, respectively. Patients with histology of inflammation showed a significantly higher CD8(+) T-cell response to five HCV-derived epitopes (YLLPRRGPRL [core], CINGVCWTV [NS3], LLCPAGHAV [NS3], ILAGYGAGV [NS4B], and GLQDCTMLV [NS5B]) as compared with patients with histology of fibrosis/cirrhosis. Also, patients with histology of inflammation showed a significantly higher CD4(+) Th1 response to the HCV core antigen as compared to patients with histology of fibrosis/cirrhosis. These results indicate that a lack of an optimal T-cell response to HCV is associated with the development of cirrhosis during chronic HCV infection.     

Hepatitis C virus (HCV)-specific memory B-cell responses in transiently and chronically infected HIV positive individuals

BackgroundAntibody responses to hepatitis C virus (HCV) occur delayed and overly decline after viral clearance indicating that the B-cell response to HCV is abnormal. Virus-specific memory B-cells have recently been found in infected individuals, but the viral exposure requirements for the generation of these cells is unknown.ObjectivesThe primary goal of this study was to quantify and compare the HCV-specific memory B-cell response between chronic and resolved HCV-infected individuals. A secondary goal was to examine if HIV-specific memory B-cell responses are maintained during HCV co-infection.Study designHCV core protein- and HIV-specific memory B-cell responses were examined in HIV/HCV-infected individuals treated 4–30 weeks after HCV diagnosis. Memory B-cell frequencies were compared between chronically and transiently infected individuals.ResultsChronically infected individuals had vigorous HCV-specific memory B-cell responses and antibodies, whereas subjects with transient viremia showed low or undetectable virus-specific B-cell responses. In addition, chronically HIV/HCV-infected subjects had robust HIV-specific memory B-cell responses.ConclusionsWhereas chronic HCV infection induces virus-specific antibodies and memory B-cells, transient infection in individuals with sustained viral response to therapy does not stimulate a durable HCV-specific B-cell response indicating that the formation of long-lived virus-specific B-cells is suppressed in the early phase of infection. This may contribute to the inability to spontaneously clear HCV 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.     

Prediction and identification-based prediction of Chinese hepatitis C viral-specific cytotoxic T lymphocyte epitopes

Cytotoxic T lymphocytes (CTLs) play a critical role in the host immune response to infection by the Hepatitis C Virus (HCV). In the current study, a number of HCV CTL epitopes that represent the HLA polymorphisms found in the majority of Chinese people were predicted based on genomic and bioinformatic approaches. The predicted epitopes were evaluated for validity by examining the peptide-binding affinity for MHC class I molecules, the stability of peptide-MHC complexes, and frequencies of IFN γ-positive T cells. Among the predicted epitope peptides, HLA-A2 restricted epitopes [NS4B (1793-1801) SMMAFSAAL] and HLA-B7 restricted epitopes [P7 (774-782) AAWYIKGRL] were able to induce high frequencies of IFN γ-producing T cells, and the specific CTLs for other epitopes were not detected in peripheral blood lymphocytes from patients with HCV. Moreover, NS4B (1793-1801) exhibited high binding affinity for HLA-A2 molecules, and its stability of peptide-MHC class I complexes was sufficient, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. Primary analyses of the immunogenicity of predicted epitopes, such as in the current study, will contribute to the future design of an efficient vaccine that will be able to induce vigorous, sustainable, and broad HCV-specific CTL responses for the Chinese population.     

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute-phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I-restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8(+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi-species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute-phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.     

Differences in HCV-specific T cell responses between chronic HCV infection and HIV/HCV co-infection

Hepatitis C virus (HCV)-specific CD4+ and CD8+ T cell responses were investigated using a panel of 728 overlapping peptides spanning the whole HCV genome in 47 HCV mono-infected and 26 HIV/HCV co-infected individuals using the IFN-gamma ELISPOT assay and flow cytometry. The frequency of HCV-specific T cell responses was similar (approximately 40%) in both groups, but the breadth of the T cell responses tended to be reduced in HIV/HCV co-infected individuals. Of interest, 23 new HCV-derived epitopes were identified, and CD4+ HCV-specific T cell responses were detected overall in a proportion similar to CD8+ T cell responses. A tendency towards a dominant CD8+ T cell response was associated with HIV/HCV co-infection. HCV-specific CD8+ T cells secreted both IL-2 and IFN-gamma, although a reduction in the percentage of IL-2/IFN-gamma-secreting cells was observed in HIV/HCV co-infected individuals. The increase in CD4+ T cell counts after antiretroviral therapy in HIV/HCV co-infected individuals was not associated with restoration of HCV-specific T cell responses. Altogether, these results provide new insights into the characterization of HCV-specific T cell responses in HCV mono-infected and HIV/HCV co-infected individuals.     

Detection of IgE antibody specific to a hepatitis C virus-derived peptide being recognized by cellular immunity in patients with HCV infection

The determination of immunogenic peptides of hepatitis C virus (HCV) is pivotal for vaccine development. We previously reported that the majority of patients infected with HCV have significant levels of IgG specific to an HCV-derived peptide at positions 35-44 of core protein (C35-44), a major epitope recognized by cellular immunity. This study addresses whether or not the other subclasses of immunoglobulins to this peptide exist. As a result, IgE, but not IgM or IgA, specific to this peptide is consistently detectable in the majority of patients with HCV infection, regardless of the different HLA types and disease conditions. These results provide additional information on this immunogenic peptide with new insights that contribute to a better understanding of host responses to HCV.     

多CTL表位DNA疫苗诱导特异性CTL应答的研究

目的:构建多CTL表位DNA疫苗诱导特异性CTL应答。方法:将编码两个HCVCTL表位(H-2d)的基因序列插入真核表达载体pcDNA3.1中,构建多CTL表位DNA疫苗。用其免疫BALB/c小鼠后,以经不同CTL抗原肽冲击的P815细胞(H-2d)作为靶细胞,进行CTL杀伤试验。结果:多CTL表位DNA疫苗可诱导机体产生针对其编码的两种HCVCTL表位的特异性CTL免疫应答,且总的特异性CTL杀伤效应增强。结论:通过天然侧翼氨基酸相连的多个CTL表位为编码序列构建的多CTL表位DNA疫苗,不但能诱导机体产生针对各个CTL表位的特异性CTL应答,而且各特异性CTL应答的联合作用可显著增强总的CTL杀伤效应。     

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.     

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.     

Modulation of the peripheral T-Cell response by CD4 mutants of hepatitis C virus: transition from a Th1 to a Th2 response

A disturbing feature of hepatitis C virus (HCV) is its long-term persistence in roughly 85% of those infected. Escape mutants may play a major role in HCV persistence. Our previous studies have identified a human leukocyte antigen DRB1*15 (HLA-DRB1*15) restricted Th1 epitope in the HCV NS3 protein, NS3(358-375), and escape variants of this epitope that may emerge under immune selection. Such variants attenuate or fail to stimulate T-cell proliferation. Here we provide data from peripheral blood mononuclear cells derived from four HLA-DRB1*15 patients chronically infected with HCV, and report that naturally occurring single amino acid substitutions in the Th1 epitope NS3(358-375) fail to stimulate proliferation, which is accompanied by a shift in cytokine secretion patterns from one characteristic of a Th1 antiviral responses to a Th2 form. Further, in one patient, we demonstrate that HCV variant peptides can effectively inhibit host polyclonal peripheral T-cell proliferation. We speculate that this phenomenon may be a factor in chronic HCV infection.     

Effective induction of type 1 cytotoxic T cell responses in mice with DNA vaccine encoding two hepatitis C virus cytotoxic T lymphocyte epitopes

The aims of this study were to explain whether a multiple cytotoxic T lymphocyte (CTL) epitope-based anti-hepatitis C virus (HCV) DNA vaccine can induce specific CTL responses to each HCV CTL epitope independently and long-term CD8(+) T cell memory responses, and to determine the cytokine secretion pattern and subtype of epitope-specific cytotoxic T cells. A multi-CTL epitope gene, which consists of two epitopes of HCV (H-2(d)-restricted HCV core(133142) and E1(315322)), was cloned into the eukaryotic expression vector pcDNA3.1. BALB/c mice (H-2(d) restricted) were vaccinated intramuscularly with this multi-CTL epitope-based DNA vaccine. The epitope-specific CTLs against target cells (P815,H-2(d) restricted) pulsed with various CTL epitope peptides were detected by lactate dehydrogenase release assay, and the precursor frequency of epitope-specific CTLs was determined by limiting dilution analysis. Cytokines (interleukin [IL]-2, IL-4, and interferon-) in culture supernatants were determined by enzyme-linked immunosorbent assay. The multi-CTL epitope-based DNA vaccine directed against two HCV CTL epitopes could induce specific CTL responses to each of the two CTL epitopes independently and long-term CD8(+) T cell memory responses. The epitope-specific cytotoxic T cells produced helper T cell type 1 cytokines. This work demonstrated that multiepitope DNA vaccination is a potential strategy to control HCV infection.     

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.     

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8

The human herpesvirus 8 (HHV-8) is a human gamma2-herpesvirus that is implicated in the development of Kaposi's sarcoma (KS), primary effusion lymphoma and Castelman's disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV-8 viral antigens. In this study, using peptide-binding motifs, we selected potential human leucocyte antigen (HLA)-A2-binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV-8 antigens, respectively. HLA-A2-binding peptides were tested for their capacity to induce CTL responses in HHV-8-negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV-8-derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16-25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59-68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease-free individuals infected by HHV-8 demonstrating that the two epitopes are relevant targets of CTL-mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV-8-associated malignancies.     

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.